Polymerases, compositions, and methods of use

Abstract

Presented herein are engineered polymerase enzymes for improved incorporation of nucleotides and nucleotide analogs, particularly engineered polymerases that maintain low error rates, low phasing rates, or increased incorporation rates for second-generation fully functional nucleotides (ffNs) under reduced incorporation times, as well as methods and kits using the same.

Description

[Technical Field]

[0001] Continuing Application Data This application claims the benefit of U.S. Provisional Patent Application No. 63 / 412,241, filed September 30, 2022, and U.S. Provisional Patent Application No. 63 / 433,971, filed December 20, 2022, each of which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically to the U.S. Patent and Trademark Office via EFS-Web as an XML file entitled "0531.00238US01.xml," which is 53,163 kilobytes in size and created on September 5, 2023. The information contained in the Sequence Listing is incorporated herein by reference.

[0003] The present disclosure relates, inter alia, to modified polymerases for use in performing nucleotide incorporation reactions, particularly in the context of nucleic acid sequencing by synthesis. [Background technology]

[0004] Sequencing by synthesis (SBS) technology relies on DNA polymerase and modified nucleotides as components of the sequencing process. The modified nucleotide typically contains a 3'-hydroxy protecting group (also called a blocking group) that prevents the polymerase from incorporating additional nucleotides into the polynucleotide chain after the base on the nucleotide is added by the DNA polymerase. After determining the identity of the added base, the 3'-hydroxy protecting group is removed, and the next modified nucleotide is added by the polymerase. Useful protecting groups prevent additional nucleotides from being added to the polynucleotide chain and are reversible, e.g., easily removable from the sugar moiety of the nucleotide without damaging the polynucleotide chain. Furthermore, modified nucleotides must be compatible with the polymerase used to incorporate them into the polynucleotide chain. Summary of the Invention

[0005] The present disclosure provides polymerases with improved incorporation rates of modified nucleotides, for example, as described in U.S. Patent No. 11,293,061. These polymerases, when tested under fast cycle time SBS conditions with the second-generation fully functional nucleotides described herein, exhibit improved sequencing performance over enzymes currently used in SBS.

[0006] In one embodiment, the modified archaeal family B DNA polymerase comprises an amino acid substitution mutation at a position functionally equivalent to an amino acid in a reference archaeal family B DNA polymerase of SEQ ID NO: 1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO: 1, and the modified archaeal family B DNA polymerase comprises an amino acid substitution mutation at position Arg58 to Leu, an amino acid substitution mutation at position Tyr261 to Gly, an amino acid substitution mutation at position Asn269 to Gly or Val, an amino acid substitution mutation at position Phe283 to Gly, an amino acid substitution mutation at position Phe303 to Gly, an amino acid substitution mutation at position Phe304 to Gly, an amino acid substitution mutation at position Phe305 to Gly, an amino acid substitution mutation at position Phe306 to Gly, an amino acid substitution mutation at position Phe307 to Gly, an amino acid substitution mutation at position Phe308 to Gly, an amino acid substitution mutation at position Phe309 ... Amino acid substitution mutation to Ile or Lys, amino acid substitution mutation to Asp at Pro328, amino acid substitution mutation to Thr at Met329, amino acid substitution mutation to Ser at Gln332, amino acid substitution mutation to HisIle at Leu333, amino acid substitution mutation to Asp, Arg, Glu, Val, or Thr at Ser347, amino acid substitution mutation to Met or Ala at Asn399, amino acid substitution mutation to Met, Lys, or Gln at Phe405 amino acid substitution mutation to Met at position 406 of Arg, amino acid substitution mutation to Val at position 410 of Ile, amino acid substitution mutation to Val at position 412 of Ile, amino acid substitution mutation to Gly at position 458 of Glu, amino acid substitution mutation to Thr or Asp at position 459 of Glu, amino acid substitution mutation to Trp at position 461 of Gln, amino acid substitution mutation to Glu at position 469 of Ala, amino acid substitution mutation to Ile at position 481 of Tyr, amino acid substitution mutation to Asn or Gly or an amino acid substitution mutation to Met or Gln or Ser or Thr, an amino acid substitution mutation to Leu at Ile position 486, an amino acid substitution mutation to Asn at Trp position 504, an amino acid substitution mutation to His or Pro at Lys position 507, an amino acid substitution mutation to Lys or Met or Arg at Glu position 511, an amino acid substitution mutation to Ile or Lys or Met or Gln or Leu at Trp position 516, an amino acid substitution mutation to Ala at Tyr position 520,It is possible to incorporate an amino acid substitution mutation such as an amino acid substitution mutation of Ile at position 521 to Thr, an amino acid substitution mutation of Met at position 523 to Ile or Thr, an amino acid substitution mutation of Arg at position 526 to Asn, an amino acid substitution mutation of Ile at position 527 to Glu, an amino acid substitution mutation of Leu at position 528 to Thr, an amino acid substitution mutation of Ile at position 567 to Leu, an amino acid substitution mutation of Asn at position 568 to Gln, an amino acid substitution mutation of Leu at position 571 to Phe, Ile, Met, or Trp, an amino acid substitution mutation of Leu at position 576, an amino acid substitution mutation of Ile, Lys, Gln, Arg, Val, or Met at position 580, an amino acid substitution mutation of Lys at position Thr590, an amino acid substitution mutation of Met at position 595 Ala, or an amino acid substitution mutation of Asp at position 603 Ile. The present disclosure also provides modified archaeal Family B DNA polymerases comprising at least two amino acid substitution mutations, at least three amino acid substitution mutations, at least four amino acid substitution mutations, or at least five amino acid substitution mutations.

[0007] Also provided herein are modified Family B DNA polymerases. In one embodiment, the modified Family B DNA polymerase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 and one or more substitution mutations as described herein. In another embodiment, the modified Family B DNA polymerase is a modified archaeal Family B DNA polymerase that comprises one or more substitution mutations as described herein. The one or more substitution mutations include, but are not limited to, an amino acid substitution mutation at a position functionally equivalent to Glu580 in the reference archaeal family B DNA polymerase of SEQ ID NO: 1; an amino acid substitution mutation at a position functionally equivalent to Phe405 and Val485 in the reference archaeal family B DNA polymerase of SEQ ID NO: 1; an amino acid substitution mutation at a position functionally equivalent to Phe405 and Ile410 in the reference archaeal family B DNA polymerase of SEQ ID NO: 1; an amino acid substitution mutation at a position functionally equivalent to Phe140 and Ser407 in the reference archaeal family B DNA polymerase of SEQ ID NO: 1; an amino acid substitution mutation at a position functionally equivalent to Leu403, Ala408, Ile410, and Gly497 in the reference archaeal family B DNA polymerase of SEQ ID NO: 1; and amino acid substitution mutations at positions functionally equivalent to Phe405, Ile410, Ile412, Thr514, and Ile521 in the DNA polymerase, or at positions functionally equivalent to Phe405, Ala408, Ile410, Ile412, Thr514, and Ile521 in the reference archaeal family B DNA polymerase of SEQ ID NO: 1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group (i) with a lower error rate, (ii) with a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO: 1. In one embodiment, Terms used herein will be understood to have their ordinary meaning in the relevant art unless otherwise specified. Some terms used herein and their meanings are described below.

[0008] The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0009] The words "preferred" and "preferably" refer to embodiments of the invention that may offer certain benefits, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0010] The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0011] As used herein, where words such as "include," "includes," or "including" are used herein, it is understood that analogous embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0012] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0013] Conditions that are "suitable" for an event to occur, or "suitable" conditions, are conditions that do not prevent such an event from occurring. Thus, these conditions enable, enhance, facilitate, and / or are conducive to the event.

[0014] As used herein, "providing" in the context of a polymerase or composition means making the polymerase or composition, purchasing the polymerase or composition, or obtaining the polymerase or composition.

[0015] As used herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0016] References to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., mean that the particular feature, structure, composition, or characteristic described in connection with this embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases in various places throughout this specification do not necessarily refer to the same embodiment of the present disclosure. Furthermore, the particular features, structures, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0017] The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The following description more particularly exemplifies exemplary embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each example, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief explanation of the drawings]

[0018] The following detailed description of the exemplary embodiments of the present disclosure can be best understood when read in conjunction with the following drawings. [Figure 1-A] Schematic diagram showing alignment of polymerase amino acid sequences from Thermococcus sp. 9°N-7 (9°N, SEQ ID NO: 9), Thermococcus litoralis (Vent, SEQ ID NO: 10 and Deep Vent, SEQ ID NO: 11), Thermococcus waiotapuensis (Twa, SEQ ID NO: 12), Thermococcus kodakarensis (KOD, SEQ ID NO: 13), Pyrococcus furiosus (Pfu, SEQ ID NO: 14), and Pyrococcus abyssi (Pab, SEQ ID NO: 15). An asterisk (*) indicates a position with a single, completely conserved residue among all polymerases. A ":" (colon) indicates conservation between groups of very high similarity - roughly equivalent to a scoring above 0.5 on the Gonnet PAM 250 matrix. A "." (period) indicates conservation between groups of weak similarity - roughly equivalent to a scoring of =<0.5 and >0 on the Gonnet PAM 250 matrix. [Figure 1-B]Schematic diagram showing alignment of polymerase amino acid sequences from Thermococcus sp. 9°N-7 (9°N, SEQ ID NO: 9), Thermococcus litoralis (Vent, SEQ ID NO: 10 and Deep Vent, SEQ ID NO: 11), Thermococcus waiotapuensis (Twa, SEQ ID NO: 12), Thermococcus kodakarensis (KOD, SEQ ID NO: 13), Pyrococcus furiosus (Pfu, SEQ ID NO: 14), and Pyrococcus abyssi (Pab, SEQ ID NO: 15). An asterisk (*) indicates a position with a single, completely conserved residue among all polymerases. A ":" (colon) indicates conservation between groups of very high similarity - roughly equivalent to a scoring above 0.5 on the Gonnet PAM 250 matrix. A "." (period) indicates conservation between groups of weak similarity - roughly equivalent to a scoring of =<0.5 and >0 on the Gonnet PAM 250 matrix. [Figure 1-C]Schematic diagram showing alignment of polymerase amino acid sequences from Thermococcus sp. 9°N-7 (9°N, SEQ ID NO: 9), Thermococcus litoralis (Vent, SEQ ID NO: 10 and Deep Vent, SEQ ID NO: 11), Thermococcus waiotapuensis (Twa, SEQ ID NO: 12), Thermococcus kodakarensis (KOD, SEQ ID NO: 13), Pyrococcus furiosus (Pfu, SEQ ID NO: 14), and Pyrococcus abyssi (Pab, SEQ ID NO: 15). An asterisk (*) indicates a position with a single, completely conserved residue among all polymerases. A ":" (colon) indicates conservation between groups of very high similarity - roughly equivalent to a scoring above 0.5 on the Gonnet PAM 250 matrix. A "." (period) indicates conservation between groups of weak similarity - roughly equivalent to a scoring of =<0.5 and >0 on the Gonnet PAM 250 matrix. [Figure 1-D]Schematic diagram showing alignment of polymerase amino acid sequences from Thermococcus sp. 9°N-7 (9°N, SEQ ID NO: 9), Thermococcus litoralis (Vent, SEQ ID NO: 10 and Deep Vent, SEQ ID NO: 11), Thermococcus waiotapuensis (Twa, SEQ ID NO: 12), Thermococcus kodakarensis (KOD, SEQ ID NO: 13), Pyrococcus furiosus (Pfu, SEQ ID NO: 14), and Pyrococcus abyssi (Pab, SEQ ID NO: 15). An asterisk (*) indicates a position with a single, completely conserved residue among all polymerases. A ":" (colon) indicates conservation between groups of very high similarity - roughly equivalent to a scoring above 0.5 on the Gonnet PAM 250 matrix. A "." (period) indicates conservation between groups of weak similarity - roughly equivalent to a scoring of =<0.5 and >0 on the Gonnet PAM 250 matrix. [Figure 1-E]Schematic diagram showing alignment of polymerase amino acid sequences from Thermococcus sp. 9°N-7 (9°N, SEQ ID NO: 9), Thermococcus litoralis (Vent, SEQ ID NO: 10 and Deep Vent, SEQ ID NO: 11), Thermococcus waiotapuensis (Twa, SEQ ID NO: 12), Thermococcus kodakarensis (KOD, SEQ ID NO: 13), Pyrococcus furiosus (Pfu, SEQ ID NO: 14), and Pyrococcus abyssi (Pab, SEQ ID NO: 15). An asterisk (*) indicates a position with a single, completely conserved residue among all polymerases. A ":" (colon) indicates conservation between groups of very high similarity - roughly equivalent to a scoring above 0.5 on the Gonnet PAM 250 matrix. A "." (period) indicates conservation between groups of weak similarity - roughly equivalent to a scoring of =<0.5 and >0 on the Gonnet PAM 250 matrix. [Figure 2A] Some exemplary mutations considered for secondary screening are shown. [Figure 2B] Some exemplary mutations considered for secondary screening are shown. [Figure 3] The performance of Tier 1 mutations obtained from the secondary screen is shown. [Figure 4A] Tier 2 mutation performance obtained from secondary screening is shown. [Figure 4B] Tier 2 mutation performance obtained from secondary screening is shown. [Figure 5] An example of double mutation performance during secondary screening is shown. [Figure 6] Examples of polymerases tested (polymerases A, B, E, F, H, I, J, L, N, and O) in a 150-cycle run with short incubation times (20 seconds) are shown. [Figure 7]1 shows a comparison of polymerase performance in a 150-cycle run with short integration times. [Figure 8-1] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-2] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-3] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-4] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-5] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-6] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-7] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-8] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-9] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-10] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-11] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-12] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-13] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-14] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-15] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown. [Figure 8-16] The amino acid sequences of SEQ ID NOs: 1 to 32 are shown.

[0019] The schematic diagrams are not necessarily drawn to scale. Like numbers used in the figures refer to like components, steps, etc. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. Furthermore, the use of different numbers to refer to a component is not intended to indicate that the differently numbered component may not be the same as or similar to the other numbered component. DETAILED DESCRIPTION OF THE INVENTION

[0020] polymerase Provided herein are polymerases, compositions and kits comprising the polymerases, and methods for using the polymerases. The polymerases described herein are deoxyribonucleic acid (DNA) polymerases. In one embodiment, the polymerases of the present disclosure, also referred to herein as "altered polymerases," are based on the amino acid sequence of a reference polymerase. The altered polymerases contain substitution mutations at one or more residues compared to the reference polymerase. The substitution mutations can be at the same position or at functionally equivalent positions compared to the reference polymerase. The reference polymerases and functionally equivalent positions are described herein. One of skill in the art will readily understand that the altered polymerases of the present disclosure do not exist in nature.

[0021] The modified polymerases described herein have useful activity compared to reference polymerases. The reference polymerases described herein can be used in SBS reactions with modified nucleotides having a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group. Modified nucleotides having a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group are referred to herein as "second-generation fully functional nucleotides" or "second-generation ffNs." In one or more embodiments, second-generation ffNs can also include a cleavable linker. Second-generation ffNs are described herein. Unless otherwise specified, references herein to the incorporation of a nucleotide by a polymerase refer to the incorporation of a second-generation ffN.

[0022] The reference polymerases described herein have useful error rates in SBS reactions when using second-generation ffN and standard incorporation rates. However, using a reference polymerase in an SBS reaction with a fast incorporation time increases the error rate. Maintaining or exceeding current levels of performance at faster incorporation times can be assisted by new-generation polymerases. Presented herein are DNA polymerases with significantly improved performance under fast cycle time SBS conditions using second-generation ffN. The inventors surprisingly identified specific modified polymerases that exhibit improved characteristics, including improved accuracy, during fast incorporation times using second-generation ffN. Improved accuracy includes reduced error rate and reduced phasing, resulting in improved quality metrics in SBS reactions. Thus, the modified polymerases of the present disclosure have one or more activities selected from a lower error rate, a lower phasing rate, and / or an increased incorporation rate of second-generation ffN compared to a reference sequence, such as reference sequence SEQ ID NO: 1.

[0023] "Error rate" refers to a measure of the frequency of errors in identifying the correct base (i.e., the complement of the template sequence at a particular position) during a call on a cluster during a sequencing reaction. The fidelity with which a sequenced library matches the original genome sequence can vary depending on the frequency of base mutations that occur at any stage from nucleic acid extraction to its sequencing on the sequencing platform. This frequency places an upper limit on the probability that the sequenced base is correct. In some embodiments, the quality score is presented as a numerical value. For example, a quality score may be quoted as QXX, where XX is the score, which indicates the probability that that particular call is 10 -XX / 10 Thus, as an example, Q30 equates to an error rate of 1 in 1000, or 0.1%, and Q40 equates to an error rate of 1 in 10,000, or 0.01%.

[0024] Phasing is a term well known to those skilled in the art and is used to describe the loss of synchronization in the reading of sequence copies of a cluster. Phasing causes the extracted intensity for a particular cycle to include the signal of the current cycle and noise from the previous cycle. Therefore, as used herein, the term "phasing" refers to a phenomenon in SBS caused by the incomplete incorporation of nucleotides into some parts of the DNA strand within a cluster by the polymerase in a given sequencing cycle, and is therefore a measure of the rate at which single molecules within a cluster lose synchronization with each other. Phasing can be measured during the detection of cluster signals in each cycle and reported as the percentage of detectable signals from the cluster that are out of synchronization with the signals within the cluster. As an example, a cluster is detected by a "green" fluorophore signal during cycle N. In the next cycle (cycle N+1), 99.9% of the cluster signal is detected in the "red" channel, and 0.1% of the signal remains from the previous cycle and is detected in the "green" channel. The result indicates that fading is occurring and can be reported as a numerical value, such as a fading value of 0.1, indicating that 0.1% of the molecules in the cluster are delayed with each cycle.

[0025] Detection of phasing can be performed and reported according to any suitable method known in the art, for example, as described in U.S. Patent No. 8,965,076 and U.S. Provisional Patent No. 62 / 535558. For example, as described in the Examples below, phasing is routinely detected and reported during SBS sequencing runs on sequencing instruments such as the HiSeq™, Genome Analyzer™, NextSeq™, NextSeq1000™, NextSeq2000™, NovaSeq™, iSeq™, MiniSeq™, or MiSeq™ sequencing platforms from Illumina, Inc. (San Diego, California), or any other suitable instrument known in the art.

[0026] Reduced cycle time can increase the occurrence of phasing, which contributes to error rate. The discovery of the mutations described herein in the modified polymerase that reduce the occurrence of phasing when used under fast cycle time conditions with second-generation ffN is unexpected and provides significant advantages in SBS applications. For example, when using second-generation fFN, the modified polymerase can provide faster SBS cycle time with lower phasing, and optionally longer sequencing read length. The error rate and phasing characteristics of the modified polymerases provided herein are described in the Examples section below.

[0027] In one embodiment, when the modified polymerase is tested using a fast incorporation time, a reduced error rate occurs compared to enzymes currently used in SBS, such as SEQ ID NO: 1. Incorporation refers to the amount of time the DNA polymerase is in contact with the template. As used herein, slow incorporation times are those used under standard cycling using a MiniSeq™ benchtop sequencing system. Slow incorporation times include 40 seconds to 50 seconds. As used herein, fast cycle times refer to incorporation steps that are between 10 seconds and 40 seconds. In one embodiment, SBS fast cycle time conditions are incorporation times of 40 seconds or less, 30 seconds or less, 20 seconds or less, 18 seconds or less, 16 seconds or less, 14 seconds or less, 12 seconds or less, or 10 seconds or less. In one embodiment, SBS fast cycle time conditions are incorporation times of at least 10 seconds, at least 12 seconds, at least 14 seconds, at least 16 seconds, at least 18 seconds, at least 20 seconds, or at least 30 seconds.

[0028] The modified polymerases described herein can be used in SBS reactions for runs of different lengths. A "run" refers to the number of nucleotides identified on a template. A run typically includes a run based on a first primer (e.g., a read 1 primer) that reads one strand of the template and a run based on a second primer (e.g., a read 2 primer) that reads the complementary strand of the template. In one embodiment, the number of nucleotides identified using the first or second primer can be 10 to 300 nucleotides. In one embodiment, the number of nucleotides identified using the first or second primer can be 300 nucleotides or less, 250 nucleotides or less, 200 nucleotides or less, 150 nucleotides or less, 150 nucleotides or less, 130 nucleotides or less, 110 nucleotides or less, 90 nucleotides or less, 70 nucleotides or less, 50 nucleotides or less, 30 nucleotides or less, or 20 nucleotides or less. In one embodiment, the number of nucleotides identified using the first primer or the second primer can be at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 50 nucleotides, at least 70 nucleotides, at least 90 nucleotides, at least 110 nucleotides, or at least 130 nucleotides.

[0029] In certain embodiments, the modified polymerase is based on a Family B-type DNA polymerase. The modified polymerase may be based, for example, on a Family B archaeal DNA polymerase, human DNA polymerase-α, or a phage polymerase. An modified polymerase of the present disclosure "based on" a Family B-type DNA polymerase means that the modified polymerase is a Family B-type DNA polymerase that includes one or more of the substitution mutations described herein. An modified polymerase of the present disclosure "based on" a Family B-type DNA polymerase may also include one or more conservative mutations and / or one or more non-conservative mutations, as described herein.

[0030] Family B archaeal DNA polymerases are well known in the art, as exemplified by the disclosure of U.S. Patent No. 8,460,910. In certain embodiments, the modified polymerases of the present disclosure are based on Family B archaeal DNA polymerases, are derived from hyperthermophilic archaea, and are thermostable.

[0031] In certain embodiments, the Family B archaeal DNA polymerase is from a genera such as, for example, Thermococcus, Pyrococcus, Methanococcus, Pyrobaculum, Pyrodictium, and Aeropyrum. Members of the genus Thermococcus are well known in the art and include, but are not limited to, T. 4557, T. barophilus, T. gammatolerans, T. onnurineus, T. sibiricus, T. kodakarensis, T. gorgonarius (TGO), and T. waiotapuensis. Members of the genus Pyrococcus are well known in the art and include, but are not limited to, P. NA2, P. abyssi, P. furiosus, P. horikoshii, P. yayanosii, P. endeavori, P. glycovorans, and P. woesei. Members of the genus Methanococcus are well known in the art and include, but are not limited to, M. aeolicus, M. maripaludis, M. vannielii, M. voltae, M. thermolithotrophicus, and M. jannaschii. Members of the genus Pyrobaculum are well known in the art and include, but are not limited to, P. calidifontis (Pc). Members of the genus Pyrodictium are well known in the art and include, but are not limited to, P. occultum. Members of the genus Aeropyrum are well known in the art and include, but are not limited to, A. pernix.

[0032] In one embodiment, the modified Family B DNA polymerase is based on Vent®, Deep Vent®, 9°N, Pfu, KOD, or Pab polymerase. Vent and Deep Vent are trade names used for Family B DNA polymerases isolated from the hyperthermophilic archaeon Thermococcus litoralis. 9°N polymerase is a Family B polymerase isolated from Thermococcus sp. 9°N-7. Pfu polymerase is a Family B polymerase isolated from Pyrococcus furiosus. KOD polymerase is a Family B polymerase isolated from Thermococcus kodakarensis. Pab polymerase is a Family B polymerase isolated from Pyrococcus abyssi. Twa is a Family B polymerase isolated from T. waiotapuensis. Examples of Vent® polymerase, Deep Vent® polymerase, 9°N polymerase, Pfu polymerase, KOD polymerase, Pab polymerase, and Twa polymerase are disclosed in FIG. 1.

[0033] In certain embodiments, the modified polymerase is based on a family B DNA polymerase from a phage, such as, for example, T4, RB69, or phi29 phage.

[0034] FIG. 1 shows a sequence alignment of proteins having the amino acid sequences set forth in SEQ ID NOs: 9-15. The alignment indicates amino acids that are conserved among different Family B polymerases. One skilled in the art will understand that conserved amino acids and conserved regions are most likely conserved because they are important to polymerase function and therefore indicate a correlation between polymerase structure and function. The alignment also indicates regions of variability across different Family B polymerases. From such data, one skilled in the art can infer regions of the polymerase where substitutions, particularly conservative substitutions, can be tolerated without unduly affecting the biological activity of the modified polymerase.

[0035] The modified polymerases described herein can be based on the amino acid sequence of a known polymerase (also referred to herein as a reference polymerase) and further include substitution mutations at one or more residues. In one embodiment, the substitution mutations are at functionally equivalent positions to the amino acids in the reference polymerase. By "functionally equivalent," it is meant that the modified polymerase has an amino acid substitution at an amino acid position in the reference polymerase that has the same functional role in both the reference polymerase and the modified polymerase.

[0036] Generally, functionally equivalent substitution mutations in two or more different polymerases occur at homologous amino acid positions within the amino acid sequences of the polymerases. Therefore, the use of the term "functionally equivalent" herein also encompasses mutations that are "positionally equivalent" or "homologous" to a given mutation, regardless of whether the specific function of the mutated amino acid is known. Functionally and positionally equivalent amino acid residues in the amino acid sequences of two or more different polymerases can be identified based on sequence alignment and / or molecular modeling. An example of a sequence alignment for identifying positionally and / or functionally equivalent residues is shown in Figure 1. For example, the tandemly aligned residues in Figure 1 of the Twa, KOD, Pab, Pfu, Deep Vent, and Vent polymerases are considered to be both positionally equivalent and functionally equivalent to the corresponding residues in the 9°N polymerase amino acid sequence. Thus, for example, residue 358 of 9°N polymerase, Twa polymerase, KOD polymerase, Pfu polymerase, Deep Vent polymerase, and Pab polymerase, and residue 360 ​​of Vent polymerase, are functionally equivalent and positionally equivalent. Similarly, for example, residue 633 of 9°N polymerase, Twa polymerase, KOD polymerase, and Pab polymerase, residue 634 of Pfu polymerase and Deep Vent polymerase, and residue 636 of Vent polymerase are functionally equivalent and positionally equivalent. Those skilled in the art can easily identify functionally equivalent residues in DNA polymerases.

[0037] In certain embodiments, the substitution mutation comprises a mutation to a residue having a non-polar side chain. Amino acids with non-polar side chains are well known in the art and include, for example, alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.

[0038] In certain embodiments, the substitution mutation comprises a mutation to a residue having a polar side chain. Amino acids with polar side chains are well known in the art and include, for example, arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, cysteine, tyrosine, and threonine.

[0039] In certain embodiments, the substitution mutation comprises a mutation to a residue having a hydrophobic side chain. Amino acids with hydrophobic side chains are well known in the art and include, for example, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan.

[0040] In certain embodiments, the substitution mutation comprises a mutation to a residue having an uncharged side chain. Amino acids having uncharged side chains are well known in the art and include, for example, glycine, serine, cysteine, asparagine, glutamine, tyrosine, and threonine.

[0041] In one or more embodiments, the modified polymerase has an amino acid sequence structurally similar to a reference polymerase disclosed herein. In one embodiment, the reference polymerase comprises the amino acid sequence of archaeal family B DNA polymerase 1901, also referred to herein as Pol1901 (SEQ ID NO: 1). Other reference sequences include SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, which have substitution mutations functionally equivalent to the following in SEQ ID NO: 1: Met 129 Ala, Asp 141 Ala, Glu 143 Ala, Cys 223 Ser, Arg 247 Tyr, Thr 349 Lys, Leu 408 Ala, Tyr 409 Ala, Pro 410 Ile, Ala 485 Val, Tyr 497 Gly, Glu 599 Asp, and His 633 Gly.

[0042] As used herein, an altered polymerase may be "structurally similar" to a reference polymerase if the amino acid sequence of the altered polymerase has a particular amount of sequence similarity and / or sequence identity compared to the reference polymerase.

[0043] The structural similarity of two amino acid sequences can be determined by aligning the residues of the two sequences (e.g., a candidate polymerase and a reference polymerase described herein) to optimize the number of identical amino acids along the length of the sequences; gaps in either or both sequences are allowed when aligning to optimize the number of identical amino acids, but the amino acids in each sequence must still remain in their proper order. A candidate polymerase is a polymerase that is compared to the reference polymerase. A candidate polymerase that has structural similarity and polymerase activity to the reference polymerase is a modified polymerase.

[0044] Pairwise comparative analysis of amino acid or nucleotide sequences can be performed, for example, by the homology alignment algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity method study of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (generally, Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, Greene Publishing). Associates, Inc. and John Wiley & Sons, Inc. (see supplement for 2004).

[0045] Unless otherwise modified as described herein, the algorithm used to determine structural similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., J. Mol. Biol. 215:403-410 (1990)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as possible to increase the cumulative alignment score. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value, the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM 62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0046] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90, 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0047] In comparing two amino acid sequences, structural similarity may be referred to by the percentage "identity" or by the percent "similarity." "Identity" refers to the presence of identical amino acids. "Similarity" refers not only to the presence of identical amino acids but also to the presence of conservative substitutions. Conservative substitutions for amino acids in proteins may be selected from other members of the class to which the amino acid belongs. For example, it is well known in the field of protein biochemistry that an amino acid belonging to a group of amino acids having a particular size or characteristic (e.g., charge, hydrophobicity, or hydrophilicity) can be substituted for another amino acid without altering the activity of the protein, especially in regions of the protein not directly related to biological activity. For example, nonpolar amino acids include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Hydrophobic amino acids include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. Polar amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, cysteine, tyrosine, and threonine. Uncharged amino acids include glycine, serine, cysteine, asparagine, glutamine, tyrosine, and threonine, among others.

[0048] Thus, as used herein, a reference to a polymerase described herein, for example, a reference to the amino acid sequence of one or more SEQ ID NOs described herein, can include proteins having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference polymerase.

[0049] Alternatively, as used herein, a reference to a polymerase described herein, for example, a reference to the amino acid sequence of one or more SEQ ID NOs described herein, can include proteins having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the reference polymerase.

[0050] The present disclosure describes mutations that result in polymerases with one or more of the polymerase activities described herein. The polymerases described herein can include any number of mutations, e.g., at least one, at least two, at least three, at least four, or at least five mutations, compared to a reference polymerase, such as SEQ ID NO: 1. Similarly, the polymerases described herein can include any combination of mutations.

[0051] In one or more embodiments, the modified Family B polymerases described herein include a substitution mutation at a position functionally equivalent to R58, Y261, N269, F283, P328, M329, Q332, L333, S347, N399, F405, R406, 1410, 1412, E458, E459, Q461, A469, Y481, V485, 1486, W504, K507, E511, W516, Y520, 1521, M523, R526, E527, 1567, N568, L571, E576, E580, T590, A595, or 1603 in Pol1901 (SEQ ID NO: 1).

[0052] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Arg58 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Arg58 is a mutation to a nonpolar amino acid or a hydrophobic amino acid, such as Leu.

[0053] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Tyr261 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Tyr261 is a mutation to a non-polar, hydrophobic, or uncharged amino acid, e.g., Gly.

[0054] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Asn269 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Asn269 is to a non-polar amino acid, e.g., Gly or Val. In one embodiment, the substitution mutation at the position functionally equivalent to Asn269 is to a hydrophobic amino acid, e.g., Gly or Val. In one embodiment, the substitution mutation at the position functionally equivalent to Asn269 is to an uncharged amino acid, e.g., Gly.

[0055] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Phe283 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Phe283 is to a non-polar, hydrophobic, or uncharged amino acid, e.g., Ile. In one embodiment, the substitution mutation at the position functionally equivalent to Phe283 is to a polar amino acid, e.g., Lys.

[0056] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Pro328 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Pro328 is to a polar amino acid or an uncharged amino acid, e.g., Asp.

[0057] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Met329 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Met329 is to a polar amino acid or an uncharged amino acid, e.g., Thr.

[0058] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Gln332 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Gln332 is to a polar amino acid or an uncharged amino acid, e.g., Ser.

[0059] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Leu333 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Leu333 is to a polar amino acid, e.g., His. In one embodiment, the substitution mutation at the position functionally equivalent to Leu333 is to a non-polar amino acid, e.g., He. In one embodiment, the substitution mutation at the position functionally equivalent to Leu333 is to a hydrophobic amino acid, e.g., He.

[0060] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to S347 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to S347 is to a polar amino acid, e.g., Asp, Arg, or Glu. In one embodiment, the substitution mutation at the position functionally equivalent to S347 is to a non-polar amino acid, e.g., Val. In one embodiment, the substitution mutation at the position functionally equivalent to S347 is to a hydrophobic amino acid, e.g., Val. In one embodiment, the substitution mutation at the position functionally equivalent to S347 is to a polar amino acid or an uncharged amino acid, e.g., Thr.

[0061] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Asn399 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Asn399 is to a non-polar amino acid, e.g., Ala or Met. In one embodiment, the substitution mutation at the position functionally equivalent to Asn399 is to a hydrophobic amino acid, e.g., Ala or Met.

[0062] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Phe405 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Phe405 is a mutation to a non-polar amino acid or a hydrophobic amino acid, such as Met. In one embodiment, the substitution mutation at the position functionally equivalent to Phe405 is a mutation to a polar amino acid, such as Lys or Gln. In one embodiment, the substitution mutation at the position functionally equivalent to Phe405 is a mutation to an uncharged amino acid, such as Gln. In one embodiment, the substitution mutation at the position functionally equivalent to Phe405 is a mutation to Met. In one embodiment, the substitution mutation at the position functionally equivalent to Phe405 is a mutation to Lys.

[0063] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Arg406 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Arg406 is to a nonpolar amino acid or a hydrophobic amino acid, e.g., Met.

[0064] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Ile410 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Ile410 is to a polar amino acid or an uncharged amino acid, e.g., Ser.

[0065] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Ile412 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Ile412 is a mutation to a non-polar amino acid, e.g., Val. In one embodiment, the substitution mutation at the position functionally equivalent to Ile412 is a mutation to a hydrophobic amino acid, e.g., Val.

[0066] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Glu458 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Glu458 is a mutation to a non-polar, hydrophobic, or uncharged amino acid, e.g., Gly.

[0067] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Glu459 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Glu459 is to a polar amino acid or an uncharged amino acid, e.g., Thr. In one embodiment, the substitution mutation at the position functionally equivalent to Glu459 is to a polar amino acid or an uncharged amino acid, e.g., Asp.

[0068] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Gln461 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Gln461 is a mutation to a polar amino acid, e.g., Tyr. In one embodiment, the substitution mutation at the position functionally equivalent to Gln461 is a mutation to an uncharged amino acid, e.g., Tyr.

[0069] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Ala469 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Ala469 is to a polar amino acid, e.g., Glu.

[0070] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Tyr481 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Tyr481 is to a non-polar amino acid, e.g., Ile.

[0071] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Val485 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Val485 is to a non-polar or hydrophobic amino acid, e.g., Met or Gly, preferably Gly. In one embodiment, the substitution mutation at the position functionally equivalent to Val485 is to a polar amino acid, e.g., Lys, Asn, Gln, Ser, or Thr, preferably Asn or Lys. In one embodiment, the substitution mutation at the position functionally equivalent to Val485 is to a hydrophobic amino acid, e.g., Gly. In one embodiment, the substitution mutation at the position functionally equivalent to Val485 is to an uncharged amino acid, e.g., Asn, Gly, Gln, Ser, or Thr.

[0072] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Ile486 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Ile486 is a mutation to a non-polar amino acid, e.g., Leu. In one embodiment, the substitution mutation at the position functionally equivalent to Ile486 is a mutation to a hydrophobic amino acid, e.g., Leu.

[0073] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Trp504 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Trp504 is to a polar amino acid, e.g., Asn. In one embodiment, the substitution mutation at the position functionally equivalent to Trp504 is to an uncharged amino acid, e.g., Asn.

[0074] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Lys507 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Lys507 is to a polar amino acid, e.g., His. In one embodiment, the substitution mutation at the position functionally equivalent to Lys507 is to a non-polar amino acid or a hydrophobic amino acid, e.g., Pro.

[0075] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Glu511 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Glu511 is to a polar amino acid, e.g., Arg or Lys. In one embodiment, the substitution mutation at the position functionally equivalent to Glu511 is to a non-polar amino acid, e.g., Met. In one embodiment, the substitution mutation at the position functionally equivalent to Glu511 is to a hydrophobic amino acid, e.g., Met.

[0076] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Trp516 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Trp516 is a mutation to a polar amino acid, e.g., Gln or Lys. In one embodiment, the substitution mutation at the position functionally equivalent to Trp516 is a mutation to a non-polar amino acid or a hydrophobic amino acid, e.g., Ile or Met. In one embodiment, the substitution mutation at the position functionally equivalent to Trp516 is a mutation to a hydrophobic amino acid, e.g., Ile. In one embodiment, the substitution mutation at the position functionally equivalent to Trp516 is a mutation to an uncharged amino acid, e.g., Gln. In one embodiment, the substitution mutation at the position functionally equivalent to Trp516 is a mutation to an uncharged amino acid, e.g., Leu.

[0077] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Tyr520 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Tyr520 is to a non-polar amino acid, e.g., Ala. In one embodiment, the substitution mutation at the position functionally equivalent to Tyr520 is to a hydrophobic amino acid, e.g., Ala.

[0078] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Ile521 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Ile521 is to a polar amino acid or an uncharged amino acid, e.g., Thr.

[0079] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Met523 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Met523 is to a polar amino acid or an uncharged amino acid, e.g., Thr. In one embodiment, the substitution mutation at the position functionally equivalent to Met523 is to a nonpolar amino acid or a hydrophobic amino acid, e.g., He.

[0080] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Arg526 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Arg526 is to a polar amino acid or an uncharged amino acid, such as Asn.

[0081] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Glu527 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Glu527 is a mutation to a nonpolar amino acid or a hydrophobic amino acid, e.g., Ile.

[0082] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Leu 528 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Leu 528 is to a polar amino acid or an uncharged amino acid, e.g., Thr.

[0083] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Ile567 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Ile567 is a mutation to a non-polar amino acid, e.g., Leu. In one embodiment, the substitution mutation at the position functionally equivalent to Ile567 is a mutation to a hydrophobic amino acid, e.g., Leu.

[0084] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Asn568 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Asn568 is to a polar amino acid, e.g., Gln. In one embodiment, the substitution mutation at the position functionally equivalent to Asn568 is to an uncharged amino acid, e.g., Gln.

[0085] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Leu571 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Leu571 is to a nonpolar amino acid or a hydrophobic amino acid, e.g., Phe, Ile, Met, or Trp.

[0086] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Glu576 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Glu576 is to a nonpolar amino acid or a hydrophobic amino acid, e.g., Leu.

[0087] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Glu580 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Glu580 is a mutation to a polar amino acid, e.g., Gln, Lys, or Arg, preferably Lys. In one embodiment, the substitution mutation at the position functionally equivalent to Glu580 is a mutation to a non-polar amino acid, e.g., Ile, Val, or Met. In one embodiment, the substitution mutation at the position functionally equivalent to Glu580 is a mutation to a hydrophobic amino acid, e.g., Ile or Val. In one embodiment, the substitution mutation at the position functionally equivalent to Glu580 is a mutation to an uncharged amino acid, e.g., Gln. In one embodiment, the substitution mutation at the position functionally equivalent to Glu580 is a mutation to Arg. In one embodiment, the substitution mutation at the position functionally equivalent to Glu580 is a mutation to Ile. In one embodiment, the substitution mutation at a position functionally equivalent to Glu580 is a mutation to Lys.

[0088] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Thr590 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Thr590 is to a polar amino acid, e.g., Lys.

[0089] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Ala595 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Ala595 is to a nonpolar amino acid or a hydrophobic amino acid, e.g., Met.

[0090] In one embodiment, the modified Family B polymerase comprises a substitution mutation at a position functionally equivalent to Ile603 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Ile603 is to a polar amino acid, e.g., Asp.

[0091] The present disclosure also provides modified Family B polymerases having combinations of substitution mutations, including but not limited to those described above. In one or more embodiments, the modified Family B polymerase comprises substitution mutations at at least two, three, four, five, or six positions.

[0092] In one embodiment, the modified polymerase comprises at least two substitution mutations. The first substitution mutation is at a position functionally equivalent to Glu580 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Glu580 is to a polar amino acid, e.g., Gln, Lys, or Arg, preferably Lys. The second substitution mutation can be at a position functionally equivalent to Trp516 in Pol1901, where the substitution mutation at the position functionally equivalent to Trp516 is to a nonpolar amino acid, e.g., Ile or Met, preferably Met. Alternatively, the second substitution mutation can be at a position functionally equivalent to Trp516 in Pol1901, where the substitution mutation at the position functionally equivalent to Trp516 is to a nonpolar amino acid or a hydrophobic amino acid, e.g., Leu. In one embodiment, the substitution mutation at a position functionally equivalent to Glu580 is a mutation to Lys, and the substitution mutation at a position functionally equivalent to Trp516 is a mutation to Met or Leu.

[0093] In one embodiment, the modified polymerase comprises at least two substitution mutations. The first substitution mutation is at a position functionally equivalent to Glu580 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Glu580 is to a polar amino acid, e.g., Gln, Lys, or Arg, preferably Lys. The second substitution mutation is at a position functionally equivalent to Ala408 in Pol1901, where the substitution mutation at the position functionally equivalent to Ala408 is to a polar amino acid or an uncharged amino acid, e.g., Ser. In one embodiment, the substitution mutation at the position functionally equivalent to Glu580 is to Lys, and the substitution mutation at the position functionally equivalent to Ala408 is to Ser.

[0094] In one embodiment, the modified polymerase comprises at least two substitution mutations. The first substitution mutation is at a position functionally equivalent to Phe405 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Phe405 is to a non-polar amino acid, e.g., Met. The second substitution mutation is at a position functionally equivalent to Val485 in Pol1901, where the substitution mutation at the position functionally equivalent to Val485 is to a non-polar, hydrophobic, or uncharged amino acid, e.g., Gly. In one embodiment, the substitution mutation at the position functionally equivalent to Val485 is to Met, and the substitution mutation at the position functionally equivalent to Val485 is to Gly.

[0095] In one embodiment, the modified polymerase comprises at least two substitution mutations. The first substitution mutation is at a position functionally equivalent to Leu571 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Leu571 is a mutation to a nonpolar or hydrophobic amino acid, e.g., Phe, Ile, Met, or Trp. The second substitution mutation is at a position functionally equivalent to Glu580 in Pol1901, where the substitution mutation at the position functionally equivalent to Glu580 is a mutation to a polar amino acid, e.g., Gln, Lys, or Arg. In one embodiment, the substitution mutation at the position functionally equivalent to Leu571 is a mutation to Phe, and the substitution mutation at the position functionally equivalent to Glu580 is a mutation to Lys. In another embodiment, the substitution mutation at the position functionally equivalent to Leu571 is a mutation to Met, and the substitution mutation at the position functionally equivalent to Glu580 is a mutation to Gln.

[0096] In one embodiment, the modified polymerase comprises at least two substitution mutations. The first substitution mutation is at a position functionally equivalent to Val485 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Val485 is to a polar amino acid, e.g., Lys, Asn, Gln, Ser, or Thr, or an uncharged amino acid, e.g., Asn, Gly, Gln, Ser, or Thr. The second substitution mutation is at a position functionally equivalent to Glu580 in Pol1901, where the substitution mutation at the position functionally equivalent to Glu580 is to a polar amino acid, e.g., Gln, Lys, or Arg. In one embodiment, the substitution mutation at the position functionally equivalent to Val485 is to Thr, and the substitution mutation at the position functionally equivalent to Glu580 is to Lys.

[0097] In one embodiment, the modified polymerase comprises at least two substitution mutations. The first substitution mutation is at a position functionally equivalent to Ile410 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Ile410 is a mutation to a non-polar amino acid or a hydrophobic amino acid, e.g., Val. The second substitution mutation is at a position functionally equivalent to Glu580 in Pol1901, where the substitution mutation at the position functionally equivalent to Glu580 is a mutation to a polar amino acid, e.g., Gln, Lys, or Arg. In one embodiment, the substitution mutation at the position functionally equivalent to Ile410 is a mutation to Val, and the substitution mutation at the position functionally equivalent to Glu580 is a mutation to Lys.

[0098] In one embodiment, the modified polymerase comprises at least two substitution mutations. The first substitution mutation is at a position functionally equivalent to Phe405 in Pol1901, where the substitution mutation at the position functionally equivalent to Phe405 is a mutation to a non-polar or hydrophobic amino acid, e.g., Met. The second substitution mutation is at a position functionally equivalent to Ile410 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Ile410 is a mutation to a non-polar or hydrophobic amino acid, e.g., Val. In one embodiment, the substitution mutation at the position functionally equivalent to Phe405 is a mutation to Met, and the substitution mutation at the position functionally equivalent to Ile410 is a mutation to Val.

[0099] In one embodiment, the modified polymerase comprises at least two substitution mutations. The first substitution mutation is at a position functionally equivalent to Phe140 in Pol1901, where the substitution mutation at the position functionally equivalent to Phe140 is a mutation to a non-polar amino acid or a hydrophobic amino acid, e.g., Leu. The second substitution mutation is at a position functionally equivalent to Ser407 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Ser407 is a mutation to a non-polar amino acid or a hydrophobic amino acid, e.g., Leu. In one embodiment, the substitution mutation at the position functionally equivalent to Phe140 is a mutation to Leu, and the substitution mutation at the position functionally equivalent to Ser407 is a mutation to Leu.

[0100] In one embodiment, the modified polymerase comprises at least two substitution mutations. The first substitution mutation is at a position functionally equivalent to Phe405 in Pol1901, where the substitution mutation at the position functionally equivalent to Phe405 is a mutation to a non-polar amino acid or a hydrophobic amino acid, e.g., Met. The second substitution mutation is at a position functionally equivalent to Glu580 in Pol1901, where the substitution mutation at the position functionally equivalent to Glu580 is a mutation to a polar amino acid, e.g., Gln, Lys, or Arg. In one embodiment, the substitution mutation at the position functionally equivalent to Phe405 is a mutation to Met, and the substitution mutation at the position functionally equivalent to Glu580 is a mutation to Arg.

[0101] In one embodiment, the modified polymerase comprises at least two substitution mutations. The first substitution mutation is at a position functionally equivalent to Phe405 in Pol1901, where the substitution mutation at the position functionally equivalent to Phe405 is a mutation to a non-polar amino acid or a hydrophobic amino acid, e.g., Met. The second substitution mutation is at a position functionally equivalent to Glu580 in Pol1901, where the substitution mutation at the position functionally equivalent to Glu580 is a mutation to a polar amino acid, e.g., Gln, Lys, or Arg. In one embodiment, the substitution mutation at the position functionally equivalent to Phe405 is a mutation to Met, and the substitution mutation at the position functionally equivalent to Glu580 is a mutation to Lys.

[0102] In one embodiment, the modified polymerase comprises at least three substitution mutations: a first substitution mutation at a position functionally equivalent to Glu580 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Glu580 is to a polar amino acid, e.g., Gln, Lys, or Arg, preferably Lys; a second substitution mutation at a position functionally equivalent to Ala408 in Pol1901, where the substitution mutation at the position functionally equivalent to Ala408 is to a polar amino acid or an uncharged amino acid, e.g., Ser; a third substitution mutation at a position functionally equivalent to Ile410 in Pol1901, where the substitution mutation at the position functionally equivalent to Ile410 is to a nonpolar amino acid or a hydrophobic amino acid, e.g., Val. In one embodiment, the substitution mutation at a position functionally equivalent to Glu580 is a mutation to Lys, the substitution mutation at a position functionally equivalent to Ala408 is a mutation to Ser, and the substitution mutation at a position functionally equivalent to Ile410 is a mutation to Val.

[0103] In one embodiment, the modified polymerase comprises at least three substitution mutations: a first substitution mutation at a position functionally equivalent to Phe405 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Phe405 is to a non-polar amino acid, e.g., Met; a second substitution mutation at a position functionally equivalent to Val485 in Pol1901, where the substitution mutation at the position functionally equivalent to Val485 is to a non-polar, hydrophobic, or uncharged amino acid, e.g., Gly; and a third substitution mutation at a position functionally equivalent to Ala408 in Pol1901, where the substitution mutation at the position functionally equivalent to Ala408 is to a polar or uncharged amino acid, e.g., Ser. In one embodiment, the substitution mutation at a position functionally equivalent to Phe405 is a mutation to Met, the substitution mutation at a position functionally equivalent to Val485 is a mutation to Gly, and the substitution mutation at a position functionally equivalent to Ala408 is a mutation to Ser.

[0104] In one embodiment, the modified polymerase comprises at least four substitution mutations: a first substitution mutation at a position functionally equivalent to Phe405 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Phe405 is to a non-polar amino acid, e.g., Met; a second substitution mutation at a position functionally equivalent to Val485 in Pol1901, where the substitution mutation at the position functionally equivalent to Val485 is to a non-polar, hydrophobic, or uncharged amino acid, e.g., Gly; and a third substitution mutation at a position functionally equivalent to Ala408 in Pol1901, where the substitution mutation at the position functionally equivalent to Ala408 is to a polar or uncharged amino acid, e.g., Ser. The fourth substitution mutation is at a position functionally equivalent to Ile410 in Pol1901, and the substitution mutation at the position functionally equivalent to Ile410 is a mutation to a non-polar amino acid or a hydrophobic amino acid, such as Val.

[0105] In one embodiment, the modified polymerase comprises at least four substitution mutations: a first substitution mutation at a position functionally equivalent to Leu403 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Leu403 is to a non-polar amino acid, e.g., Met; a second substitution mutation at a position functionally equivalent to Ala408 in Pol1901, where the substitution mutation at the position functionally equivalent to Ala408 is to a polar amino acid or an uncharged amino acid, e.g., Ser; and a third substitution mutation at a position functionally equivalent to Ile410 in Pol1901, where the substitution mutation at the position functionally equivalent to Ile410 is to a non-polar amino acid or a hydrophobic amino acid, e.g., Val. The fourth substitution mutation is at a position functionally equivalent to Gly497 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Gly497 is a mutation to a non-polar amino acid, e.g., Met. In one embodiment, the substitution mutation at the position functionally equivalent to Leu403 is a mutation to Met, the substitution mutation at the position functionally equivalent to Ala408 is a mutation to Ser, the substitution mutation at the position functionally equivalent to Ile410 is a mutation to Val, and the substitution mutation at the position functionally equivalent to Gly497 is a mutation to Met.

[0106] In one embodiment, the modified polymerase comprises at least five substitution mutations: a first substitution mutation at a position functionally equivalent to Phe405 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Phe405 is to a nonpolar or hydrophobic amino acid, e.g., Ile; a second substitution mutation at a position functionally equivalent to Ile410 in Pol1901, where the substitution mutation at the position functionally equivalent to Ile410 is to a nonpolar or hydrophobic amino acid, e.g., Pro; and a third substitution mutation at a position functionally equivalent to Ile412 in Pol1901, where the substitution mutation at the position functionally equivalent to Ile412 is to a nonpolar amino acid, e.g., Met. The fourth substitution mutation is at a position functionally equivalent to Thr514 in Pol1901 (SEQ ID NO: 1), and the substitution mutation at the position functionally equivalent to Thr514 is a mutation to a non-polar amino acid or a hydrophobic amino acid, for example, Ala. The fifth substitution mutation is at a position functionally equivalent to Ile521 in Pol1901 (SEQ ID NO: 1), and the substitution mutation at the position functionally equivalent to Ile521 is a mutation to a non-polar amino acid or a hydrophobic amino acid, for example, Ala. In one embodiment, the substitution mutation at the position functionally equivalent to Phe405 is a mutation to Ile, the substitution mutation at the position functionally equivalent to Ile410 is a mutation to Pro, the substitution mutation at the position functionally equivalent to Ile412 is a mutation to Met, the substitution mutation at the position functionally equivalent to Thr514 is a mutation to Ala, and the substitution mutation at the position functionally equivalent to Ile521 is a mutation to Ala.

[0107] In one embodiment, the modified polymerase comprises at least four substitution mutations. The first substitution mutation is at a position functionally equivalent to Phe405 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Phe405 is to a non-polar or hydrophobic amino acid, e.g., Leu. The second substitution mutation is at a position functionally equivalent to Ala408 in Pol1901, where the substitution mutation at the position functionally equivalent to Ala408 is to a polar or uncharged amino acid, e.g., Ser. The third substitution mutation is at a position functionally equivalent to Ile410 in Pol1901, where the substitution mutation at the position functionally equivalent to Ile410 is to a non-polar or hydrophobic amino acid, e.g., Val. The fourth substitution mutation is at a position functionally equivalent to Ile412 in Pol1901, where the substitution mutation at the position functionally equivalent to Ile412 is to a polar or uncharged amino acid, such as Thr. The fifth substitution mutation is at a position functionally equivalent to Thr514 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Thr514 is to a nonpolar or hydrophobic amino acid, such as Ala. The sixth substitution mutation is at a position functionally equivalent to Ile521 in Pol1901 (SEQ ID NO: 1), where the substitution mutation at the position functionally equivalent to Ile521 is to a nonpolar or hydrophobic amino acid, such as Ala. In one embodiment, the substitution mutation at a position functionally equivalent to Phe405 is a mutation to Leu, the substitution mutation at a position functionally equivalent to Ala408 is a mutation to Ser, the substitution mutation at a position functionally equivalent to Ile410 is a mutation to Val, the substitution mutation at a position functionally equivalent to Ile412 is a mutation to Thr, the substitution mutation at a position functionally equivalent to Thr514 is a mutation to Ala, and the substitution mutation at a position functionally equivalent to Ile521 is a mutation to Ala.

[0108] In one or more embodiments, the modified Family B polymerases of the present disclosure can include additional substitution mutations. For example, a polymerase described herein having one or more substitution mutations can also include one or more substitution mutations at a position functionally equivalent to Lys349, Ala281, Trp397, or Gly633 in Pol1901.

[0109] In one embodiment, the modified Family B polymerase optionally comprises a substitution mutation at a position functionally equivalent to Lys349 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Lys349 is to a polar amino acid or an uncharged amino acid, such as Asn or Ser.

[0110] In one embodiment, the modified Family B polymerase optionally comprises a substitution mutation at a position functionally equivalent to Ala281 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Ala281 is to a nonpolar or hydrophobic amino acid, e.g., Gly or Phe. In one embodiment, the substitution mutation at the position functionally equivalent to Ala281 is to an uncharged amino acid, e.g., Gly.

[0111] In one embodiment, the modified Family B polymerase optionally comprises a substitution mutation at a position functionally equivalent to Phe283 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Phe283 is to a polar amino acid or an uncharged amino acid, e.g., Ser.

[0112] In one embodiment, the modified Family B polymerase optionally comprises a substitution mutation at a position functionally equivalent to Trp397 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Trp397 is to a polar amino acid or an uncharged amino acid, e.g., Cys. In one embodiment, the substitution mutation at the position functionally equivalent to Trp397 is to a nonpolar amino acid or a hydrophobic amino acid, e.g., Phe.

[0113] In one embodiment, the modified Family B polymerase optionally comprises a substitution mutation at a position functionally equivalent to Gly633 in Pol1901 (SEQ ID NO: 1). In one embodiment, the substitution mutation at the position functionally equivalent to Gly633 is to a polar amino acid or an uncharged amino acid, e.g., Thr.

[0114] Specific examples of modified polymerases include the polymerases disclosed in SEQ ID NO:2 to SEQ ID NO:32.

[0115] The modified polymerases described herein may contain additional mutations known to affect polymerase activity. One such substitution mutation is at a position functionally equivalent to Arg713 in Pol1901 (SEQ ID NO: 1). Any of a variety of substitution mutations at one or more positions known to result in reduced exonuclease activity, as is well known in the art and exemplified by U.S. Patent No. 8,623,628, may be made. In one embodiment, the substitution mutation at position Arg713 is a mutation to a non-polar, hydrophobic, or uncharged amino acid, such as Gly, Met, or Ala.

[0116] In one embodiment, the modified polymerase comprises a substitution mutation at a position functionally equivalent to Arg743 or Lys705, or a combination thereof, in Pol1901 (SEQ ID NO: 1), as is well known in the art and exemplified by the disclosure of U.S. Patent No. 8,623,628. In one embodiment, the substitution mutation at position Arg743 or Lys705 is to a non-polar or hydrophobic amino acid, e.g., Ala.

[0117] Second Generation FFN The polymerases of the present disclosure can be used with modified nucleotides, which typically include a modification at the 3'-OH of the nucleotide sugar moiety, a detectable label attached to the base via a cleavable linker, or both a 3'-OH modification and a detectable label attached to the base via a cleavable linker.

[0118] In one or more embodiments, the second generation ffN described herein comprises or has the structure of formula (I):

[0119] [ka] wherein B is a nucleobase, R4 is OH, R5 is H or a 3'-OH blocking group, R6 is H, monophosphate, diphosphate, triphosphate, thiophosphate, a phosphate ester analog, a reactive phosphorus-containing group, or a hydroxy protecting group, and R7 is H or an L1-L-L2 label (U.S. Patent Application Publication No. 2021 / 0403500).

[0120] Nucleic acid bases can be purines or pyrimidines. Examples of pyrimidines include cytosine (C) and thymine (T), 5,6-dihydrouracil and 5-alkylcytosine (for example, 5-methylcytosine). Examples of purines include adenine (A) and guanine (G), and optionally substituted purine bases, such as deazapurines, adenine, 7-deazaadenine, guanine, 7-deazaguanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (for example, 7-methylguanine), theobromine, caffeine, uric acid and isoguanine.

[0121] The label is a detectable label. Useful detectable labels are well known in the art and include, but are not limited to, fluorophores, such as fluorescent dyes.

[0122] L is a cleavable linker

[0123] [ka] Each of L1 and L2 is independently an optionally present linker moiety.

[0124] In some embodiments of the cleavable linker, L, each of X and Y is O. In some other embodiments, X is S and Y is O, or X is O and Y is S. In some embodiments, each of R1a, R1b, R2, R3a, and R3b is H. In other embodiments, at least one of R1a, R1b, R2, R3a, and R3b is halogen (e.g., fluoro, chloro) or unsubstituted C1-C6 alkyl (e.g., methyl, ethyl, isopropyl, isobutyl, or t-butyl). In some such cases, each of R1a and R1b is H and at least one of R2, R3a, and R3b is unsubstituted C1-C6 alkyl or halogen (e.g., R2 is unsubstituted C1-C6 alkyl and each of R3a and R3b is H, or R2 is H and one or both of R3a and R3b is halogen or unsubstituted C1-C6 alkyl). In one embodiment, the cleavable linker L is

[0125] [ka] (the "AOL" linker part).

[0126] The wavy line indicates the attachment of the cleavable linker L to any of L1 and L2, or the attachment of the cleavable linker L to the nucleobase and / or detectable label in the absence of L1 and / or L2. In one or more embodiments, the attachment of any of L1 or L2, or the attachment of the cleavable linker L to the nucleobase when L1 and / or L2 are absent, is at the C5 position of a pyrimidine base or the C7 position of a purine base. In one or more embodiments, L1-L-L2 is

[0127] [ka] is selected from wherein Z is —O—CH—CH═CH; and n is an integer of 1, 2, 3, 4, or 5; * indicates the point of attachment of the cleavable linker to the nucleobase, **indicates the point of attachment of the cleavable linker to the detectable label.

[0128] Useful modifications at the 3'-OH of the nucleotide sugar moiety are well known in the art, such as the 3'-O-azidomethyl blocking group -CH2N3. In one or more embodiments, the polymerases disclosed herein are used with modified nucleotides bearing a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group (U.S. Pat. No. 11,293,061; U.S. Patent Application No. 17 / 748,498).

[0129] Examples of 3'-hydroxyacetal blocking groups attached to nucleotides having a deoxyribose with a removable 3'-OH blocking group include:

[0130] [ka] and those which form a covalent bond to the 3'-carbon atom. In the formula, each R 1a and R 1b are independently H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, cyano, halogen, optionally substituted phenyl, or optionally substituted aralkyl; Each R 2a and R 2b are independently H, C1-C6 alkyl, C1-C6 haloalkyl, cyano, or halogen; Alternatively, R and R together with the atoms to which they are attached form an optionally substituted 5- to 8-membered heterocyclyl group; R 3 is H, optionally substituted C2-C6 alkenyl, optionally substituted C3-C7 cycloalkenyl, optionally substituted C2-C6 alkynyl, or optionally substituted (C1-C6 alkylene)Si(R 4 )3, Each R 4are independently H, C1-C6 alkyl, or optionally substituted C6-C 10 aryl, provided that each R 1a , R 1b , R 2a , and R 2b If is H, then R 3 is not H. (All technical and scientific terms relating to 3'-hydroxyacetal blocking groups are defined in U.S. Pat. No. 11,293,061.) In one or more embodiments, the 3'-hydroxyacetal blocking group has the structure

[0131] [ka] ("AOM" 3' blocking group).

[0132] The wavy line indicates the attachment of the oxygen to the 3' carbon of the nucleotide sugar.

[0133] Examples of 3'-hydroxyacetal blocking groups attached to nucleotides having a deoxyribose with a removable 3'-OH blocking group include:

[0134] [ka] and those which form a covalent bond to the 3'-carbon atom. R 5 and R 6 are independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C8 alkoxyalkyl, optionally substituted -(CH2) m -phenyl, optionally substituted -(CH2) n -(5- or 6-membered heteroaryl), optionally substituted -(CH) k -C3-C7 carbocyclyl or optionally substituted -(CH2) p -(3- to 7-membered heterocyclyl), Alternatively, R 5 and R 6together with the atom to which they are attached form an optionally substituted 5- to 7-membered heterocyclyl; -(CH2) m -, -(CH2) n -, -(CH2) k - and -(CH2) p each - is optionally substituted, and each of m, n, k, and p is independently 0, 1, 2, 3, or 4. (All technical and scientific terms relating to 3'-hydroxythiocarbamate blocking groups are defined in U.S. Pat. No. 11,293,061.)

[0135] Non-limiting examples of second generation fFNs useful with the modified polymerases of the present disclosure include, but are not limited to, those having formula (Ia), (Ia'), (Ib), (Ic), (Ic'), or (Id):

[0136] [ka]

[0137] [ka] R4 is OH and R6 is H, monophosphate, diphosphate, triphosphate, thiophosphate, a phosphate ester analog, a reactive phosphorus-containing group, or a hydroxy protecting group.

[0138] The methods of the present disclosure include synthesizing polynucleotides using second-generation ffN (U.S. Patent Application Publication No. 2021 / 0403500, U.S. Patent Application No. 17 / 748498). Examples of polynucleotides containing nucleotides of formula (Ia') include the following structures:

[0139] [ka]

[0140] Non-limiting examples of label conjugates, such as fluorescent dye conjugates, include those having the following structure:

[0141] [ka]

[0142] [ka] wherein PG is a 3'-OH blocking group as described in U.S. Patent Application Publication No. 2021 / 0403500, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and k is 0, 1, 2, 3, 4, or 5. In one embodiment, -O-PG is AOM. In another embodiment, -O-PG is -O-azidomethyl. In one embodiment, n is 5.

[0143] Mutant polymerase In the present disclosure, various types of mutagenesis are optionally used to modify polymerases to generate mutations, for example, according to polymerase models and model predictions as discussed herein, or using random or semi-random mutation approaches. Generally, any available mutagenesis procedure can be used to create polymerase mutations. Such mutagenesis procedures optionally include selection of mutant nucleic acids and polypeptides for one or more activities of interest (e.g., lower error rates, lower phasing rates, or increased incorporation rates for second-generation ffN). Procedures that can be used include, but are not limited to, site-directed point mutagenesis, random point mutagenesis, in vitro or in vivo homologous recombination (DNA shuffling and combinatorial overlap PCR), mutagenesis using uracil-containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, degenerate PCR, double-strand break repair, and many others well known to those of skill in the art. The starting polymerase for mutation can be any of the reference polymerases described herein, including available polymerase mutations such as those identified in, for example, U.S. Patent Nos. 8,460,910, 8,623,628, 10,421,996, 9,765,309, 9,677,057, 11,104,888, and 11,001,816.

[0144] Optionally, mutagenesis can be guided by known information, e.g., sequences, sequence comparisons, physical properties, crystal structures, and / or the like, of naturally occurring polymerase molecules or known modified or mutant polymerases (e.g., using existing mutant polymerases). However, in another class of embodiments, modifications can be essentially random (e.g., as in classical or "family" DNA shuffling; see, e.g., Crameri et al. (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution," Nature 391:288-291).

[0145] Further information on mutation patterns can be found in: Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2000 ("Sambrook"), Current Protocols in Molecular Biology, FMA Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 2011) ("Ausubel"), and PCR Protocols: A Guide to Methods and Applications (edited by Innis et al.), Academic Press Inc. (1990) ("Innis"). The following publications and references provide further details on mutation formats: Arnold, Protein engineering for unusual environments, Current Opinion in Biotechnology 4:450-455 (1993); Bass et al., Mutant Trp repressor with new DNA-binding specificities, Science 242:240-245 (1988); Bordo and Argos (1991) Proposal of "Safe" Residue Substitutions in Site-directed Mutagenesis 217:721-729; Botstein and Shortle, Strategies and applications of in vitro mutagenesis, Science 229:1193-1201 (1985); Carter et al., Improved oligonucleotide site-directed mutagenesis using M13 vectors, Nucl. Acids Res.13:4431-4443 (1985), Carter, Site-directed mutagenesis, Biochem. J. 237:1-7 (1986), Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors, Methods in Enzymol. 154:382-403 (1987), Dale et al., Oligonucleotide-directed random mutagenesis using the phosphorothioate method, Methods Mol. Biol. 57:369-374 (1996), Eghtedarzadeh and Henikoff, Use of oligonucleotides to generate large deletions, Nucl. Acids Res. 14-5115 (1986), Fritz et al., Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988), Grundstrom et al., Oligonucleotide-directed mutagenesis by microscale’shot-gun’ gene synthesis, Nucl. Acids Res. 13:3305-3316 (1985), Hayes (2002) Combining Computational and Experimental Screening for rapid Optimization of Protein Properties PNAS 99(25)15926-15931, Kunkel, The efficiency of oligonucleotide directed mutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D.M.J.(ed., Springer Verlag, Berlin) (1987); Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods in Enzymol. 154, 367-382 (1987); Kramer et al., The gapped duplex DNA approach to oligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz, Oligonucleotide-directed construction of mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367 (1987); Kramer et al., Point Mismatch Repair, Cell 38:879-887 (1984). Kramer et al., Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations, Nucl. Acids Res. 16-7207 (1988). Ling et al., Approaches to DNA mutagenesis: an overview, Anal Biochem. 254(2):157-178 (1997). Lorimer and Pastan Nucleic Acids Res.23,3067-8 (1995), Mandecki, Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis, Proc. Natl. Acad. Sci. USA 83:7177-7181 (1986), Nakamaye & Eckstein, Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis, Nucl. Acids Res. 14:9679-9698 (1986), Nambiar et al., Total synthesis and cloning of a gene coding for the ribonuclease S protein, Science 223:1299-1301 (1984), Sakamar and Khorana, Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin), Nucl. Acids Res. 14:6361-6372 (1988), Sayers et al., Y-T Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. Acids Res. 16:791-802 (1988), Sayers et al., Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide, (1988) Nucl. Acids Res.16:803-814, Sieber et al., Nature Biotechnology, 19:456-460 (2001), Smith, In vitro mutagenesis, Ann. Rev. Genet. 19:423-462 (1985), Enzymol. 100:468-500 (1983), Enzymol. 154:329-350 (1987), Stemmer, Nature 370, 389-91 (1994), Taylor et al., The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13:8749-8764 (1985), Taylor et al., The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucl. Acids Res. 13:8765-8787 (1985), Wells et al., Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, Phil. Trans. R. Soc. Lond. A 317:415- -XX / 10 423 (1986), Wells et al., Cassette mutagenesis: a efficient method for generation of multiple mutations at defined sites, Gene 34:315-323 (1985). Zoller and Smith, Oligonucleotide-directed mutagenesis using M 13-derived vectors: a efficient and general procedure for production of point mutations in any DNA fragment, Nucleic Acids Res.10:6487-6500 (1982); Zoller and Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors, Methods in Enzymol. 100:468-500 (1983); Zoller and Smith, Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template, Methods in Enzymol. 154:329-350 (1987); Clackson et al. (1991) "Making antibody fragments using phage display libraries," Nature 352:624-628; Gibbs et al. (2001) "Degenerate ol. "Gogonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling," Gene 271:13-20, and Hiraga and Arnold (2003) "General method for sequence-independent site-directed chimeragenesis: J. Mol. Biol. 330:287-296. Further details on many of the above methods can be found in Methods in Enzymology Volume 154, which also describes useful controls for problem-solving regarding various mutagenesis methods.

[0146] Production and isolation of recombinant polymerases Generally, nucleic acids encoding the polymerases provided herein can be produced by cloning, recombination, in vitro synthesis, in vitro amplification, and / or other available methods, or can be obtained from commercial sources. Various recombinant methods can be used to express expression vectors encoding the polymerases provided herein. Methods for producing, expressing, and isolating expression products of recombinant nucleic acids are well known and described in the art. Numerous exemplary mutations and combinations of mutations, as well as strategies for designing desirable mutations, are described herein. Methods for creating and selecting mutations in the active site of a polymerase, including modifying steric features within or near the active site to allow improved access by modified nucleotides, can be found herein and in, for example, PCT Publication Nos. WO 2007 / 076057 and WO 2008 / 051530.

[0147] Further useful references on mutation, recombination, and in vitro nucleic acid manipulation methods (including cloning, expression, PCR, etc.) include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152, Academic Press, Inc., San Diego, CA (Berger); Kaufman et al. (2003) Handbook of Molecular and Cellular Methods in Biology and Medicine Second Edition, Ceske (ed.), CRC Press (Kaufman); The Nucleic Acid Protocols Handbook, Ralph Rapley (ed.), (2000) Cold Spring Harbor, Humana Press Inc. (Rapley); Chen et al. (ed.) PCR Cloning Protocols, Second Edition (Methods in Molecular Biology, volume 192), Humana Press; and Viljoen et al. (2005) Molecular Diagnostic PCR Handbook, Springer, ISBN The number is 1402034032.

[0148] Additionally, many kits are commercially available for purifying plasmids or other related nucleic acids from cells (see, e.g., Stratagene's StrataClean® and Qiagen's QIAprep®). Any isolated and / or purified nucleic acid can be further manipulated to generate other nucleic acids used to transfect cells, incorporated into related vectors, infect organisms for expression, and / or otherwise. Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulating expression of the specific target nucleic acid. Vectors optionally contain a generic expression cassette containing at least one independent terminator sequence, sequences allowing replication of the cassette in eukaryotes or prokaryotes, or both (e.g., shuttle vectors), and a selectable marker in both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both.

[0149] Other useful references, for example, for cell isolation and culture (e.g., subsequent nucleic acid isolation), include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and references cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley & Sons, New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture, Fundamental Methods Springer Lab Manual, Springer-Verlag, Berlin, Heidelberg, NY; and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla. Construction of vectors containing nucleic acids encoding the modified polymerases described herein uses standard ligation techniques well known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989), or Ausubel, RM, ed., Current Protocols in Molecular Biology (1994).

[0150] The present disclosure also includes nucleic acids encoding the modified polymerases disclosed herein. A particular amino acid can be encoded by multiple codons, and particular translation systems (e.g., prokaryotic or eukaryotic cells) often exhibit codon bias; for example, different organisms often prefer one of several synonymous codons to encode the same amino acid. Thus, the nucleic acids provided herein are optionally "codon optimized," meaning they are synthesized to contain codons preferred by the particular translation system used to express the polymerase. For example, if it is desired to express a polymerase in a bacterial cell (or even a particular strain of bacteria), the nucleic acid can be synthesized to contain the codons most frequently found in the genome of that bacterial cell for efficient expression of the polymerase. A similar strategy can be used when it is desired to express a polymerase in a eukaryotic cell; for example, the nucleic acid can contain codons preferred by the eukaryotic cell.

[0151] A variety of protein isolation and detection methods are well known and can be used, for example, to isolate the polymerases from recombinant cultures of cells expressing the recombinant polymerases presented herein. For example, R. Scopes, Protein Purification, Springer-Verlag, New York, (1982); Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc., New York, (1990); Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd Edition, Wiley-Liss, New York; Walker (1996) The Protein Protocols Handbook, Humana Press, NJ; Harris and Angal (1990) Protein Purification Applications: A Practical Approach, IRL Press at Oxford, Oxford, UK; Harris and Angal Protein Purification Methods: A Practical Approach, IRL Press at Oxford, Oxford, UK; Scopes (1993) Protein Purification: Principles and Practice, 3rd Edition, Springer Verlag, New York; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition Wiley-VCH, New York; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ, and references cited therein.Further details regarding protein purification and detection methods can be found in Satinder Ahuja (ed.), Handbook of Bioseparations, Academic Press (2000).

[0152] How to use The modified polymerases provided herein can be used in sequencing procedures (e.g., sequencing-by-synthesis (SBS) techniques). Briefly, SBS can be initiated by contacting a target nucleic acid with one or more nucleotides (e.g., labeled, synthetic, modified, or a combination thereof), a DNA polymerase, etc. These features allow a primer to be extended using the target nucleic acid as a template, incorporating a labeled nucleotide that can be detected. Use of the modified polymerases described herein can result in lower error rates, lower phasing rates, lower prephasing, or increased incorporation rates (i.e., reduced incorporation times) in sequencing runs. In one or more embodiments, the labeled nucleotide can be modified to further include, for example, a reversible termination feature that terminates further primer extension once the nucleotide is added to the primer. For example, a modified nucleotide with a reversible terminator moiety can be added to a primer so that further extension does not occur until a deblocking agent is delivered to remove the moiety. In one or more embodiments, the reversible terminator moiety comprises a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group. Thus, in embodiments using reversible terminators, a deblocking reagent can be delivered to the flow cell (before or after detection occurs). Washing can be performed between the various delivery steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluidic systems, and detection platforms that can be readily adapted for use with arrays produced by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), PCT International Publication Nos. WO 04 / 018497, WO 91 / 06678, WO 07 / 123744, U.S. Patent Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, 7,405,281, and 8,343,746.

[0153] Other sequencing procedures using cyclic reactions can be used, such as pyrosequencing, which detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent nucleic acid chain (Ronaghi et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi et al., Science 281(5375), 363 (1998); U.S. Patent Nos. 6,210,891, 6,258,568, and 6,274,320). In pyrosequencing, the released PPi can be detected by its conversion to adenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATP can be detected by luciferase-generated photons. Therefore, the sequencing reaction can be monitored via a luminescence detection system. The excitation radiation source used in fluorescence-based detection systems is not required for pyrosequencing procedures. Useful fluidic systems, detectors, and procedures that can be used to apply pyrosequencing to the arrays of the present disclosure are described, for example, in PCT International Publication No. WO 2012 / 058096, U.S. Patent Application Publication No. 2005 / 0191698(A1), U.S. Patent Nos. 7,595,883, and 7,244,559.

[0154] In some embodiments, methods involving real-time monitoring of DNA polymerase activity can be used. For example, nucleotide incorporation can be detected via fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and a γ-phosphate-labeled nucleotide, or using a zero-mode waveguide. Techniques and reagents for FRET-based sequencing are described, for example, in Levene et al., Science 299, 682-686 (2003); Lundquist et al., Opt. Lett. 33, 1026-1028 (2008); and Korlach et al., Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008).

[0155] Some SBS embodiments involve detecting protons released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons may use commercially available electrical detectors and related technology from Ion Torrent (Thermo Fisher Scientific), or the sequencing methods and systems described in U.S. Patent Nos. 8,262,900, 7,948,015, 8,349,167, and U.S. Published Patent Application No. 2010 / 0137143(A1).

[0156] Thus, provided herein are methods for incorporating modified nucleotides into DNA, comprising interacting the following components: (i) a modified polymerase described herein, (ii) a DNA template, and (iii) a nucleotide solution. In certain embodiments, the DNA template can be associated with an array, including a clustered array. The DNA template can be double-stranded or single-stranded. In one or more embodiments, the nucleotide is a modified nucleotide, such as a second-generation ffN described herein. The modified nucleotide typically includes a modification at the 3'-OH of the nucleotide sugar moiety, a detectable label attached to the base via a cleavable linker, or both a 3'-OH modification and a detectable label attached to the base via a cleavable linker.

[0157] Nucleic acid encoding the modified polymerase The present disclosure also includes nucleic acid molecules encoding the modified polymerases described herein. For any modified polymerase that is a mutant of a polymerase for which the amino acid sequence encoding the polymerase, preferably the wild-type nucleotide sequence, is known, the nucleotide sequence encoding the mutant can be obtained using basic principles of molecular biology. For example, given that the wild-type nucleotide sequence encoding 9°N polymerase is known, it is possible to deduce the nucleotide sequence encoding any mutant 9°N with one or more amino acid substitutions using the standard genetic code. Similarly, nucleotide sequences can be easily derived for mutant versions of other polymerases (e.g., Vent® polymerase, Deep Vent® polymerase, Pfu polymerase, KOD polymerase, Pab polymerase, etc.). A nucleic acid molecule having the required nucleotide sequence can then be constructed using standard molecular biology techniques well known in the art.

[0158] According to the embodiments provided herein, the defined nucleic acids include not only identical nucleic acids but also any minor base variations, including substitutions resulting in synonymous codons (different codons that specify the same amino acid residue) due to degenerate coding, particularly for conservative amino acid substitutions. The class of nucleotide sequences encoding the modified polymerases disclosed herein is large but finite, and the nucleotide sequence of each member of this class can be readily determined by reference to the standard genetic code.

[0159] The term "nucleic acid sequence" also includes the complementary sequence to a given single-stranded sequence in terms of base variations, and the corresponding RNA sequence.

[0160] The nucleic acid molecules described herein may also advantageously be included in a suitable expression vector for expressing the altered polymerase protein encoded therein in a suitable host. The incorporation of cloned DNA into a suitable expression vector for subsequent transformation of the cells and subsequent selection of transformed cells is well known to those skilled in the art, as provided in Sambrook et al. (1989), Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory.

[0161] Such expression vectors include vectors having a nucleic acid according to the embodiments presented herein operably linked to regulatory sequences, such as promoter regions, capable of effecting expression of that DNA fragment. The term "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. Such vectors may be transformed into suitable host cells to provide for expression of proteins according to the embodiments presented herein.

[0162] The nucleic acid molecule may encode a protein having a prosequence, including one that encodes a leader sequence on the mature protein or preprotein, which is then cleaved by the host cell to form the mature protein. The vector may be, for example, a plasmid, virus, or phage vector provided with an origin of replication, optionally a promoter for expression of the nucleotide, and optionally a regulator of the promoter. The vector may contain one or more selectable markers, such as, for example, an antibiotic resistance gene.

[0163] Regulatory elements required for expression include a promoter sequence for binding RNA polymerase and directing an appropriate level of transcription initiation and also a translation initiation sequence for ribosome binding. For example, a bacterial expression vector can contain a promoter such as the lac promoter, and a Shine-Dalgarno sequence and the start codon AUG for translation initiation. Similarly, a eukaryotic expression vector can contain a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors can be commercially obtained or can be assembled from the described sequences by methods well known in the art.

[0164] Transcription of the DNA encoding the polymerase by higher eukaryotes can be optimized by including an enhancer sequence in the vector. Enhancers are cis-acting elements of DNA that act on a promoter to increase transcription levels. Vectors also generally contain an origin of replication as well as a selectable marker.

[0165] kit The present disclosure also provides a kit for performing a nucleotide incorporation reaction. The kit includes at least one modified polymerase described herein and a nucleotide solution in a suitable packaging material in an amount sufficient for at least one nucleotide incorporation reaction. Optionally, other reagents, such as buffers and solutions necessary for using the modified polymerase and nucleotide solution, are also included. Examples of other reagents include deblocking reagents, palladium catalysts, palladium scavengers, etc., suitable for use with second-generation fFNs (U.S. Patent Application Publication No. 2021 / 0403500, U.S. Patent Application No. 17 / 748498). Typically, instructions for use of the packaged components may be included.

[0166] In certain embodiments, the nucleotide solution comprises a labeled nucleotide. In certain embodiments, the nucleotide is a synthetic nucleotide. In certain embodiments, the nucleotide is a modified nucleotide, such as a second-generation ffN. In certain embodiments, the modified nucleotide is modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group. In certain embodiments, the modified nucleotide comprises a modified nucleotide molecule comprising a purine or pyrimidine base and a deoxyribose sugar moiety to which a removable 3'-OH blocking group is covalently attached. In one or more embodiments, the 3' carbon atom is bound to a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group. 3'-OH acetal blocking groups and 3'-OH thiocarbamate blocking groups are described herein. In one or more embodiments, the 3'-hydroxyacetal blocking group has the following structure:

[0167] [ka]

[0168] In certain embodiments, the modified nucleotides are fluorescently labeled to enable their detection. In certain embodiments, the modified nucleotides comprise a nucleotide having a base linked to a detectable label via a cleavable linker. In certain embodiments, the detectable label comprises a fluorescent label. In certain embodiments, the cleavable linker has the following structure:

[0169] [ka]

[0170] As used herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit. The packaging material is preferably constructed by well-known methods to provide a sterile, contaminant-free environment. The packaging material bears a label indicating that the components can be used to perform a nucleotide incorporation reaction. Additionally, the packaging material may include instructions indicating how to employ the materials in the kit to perform a nucleotide incorporation reaction. As used herein, the term "package" refers to a solid matrix or material, such as glass, plastic, paper, or foil, capable of retaining a polypeptide within certain limits. "Instructions for use" typically include specific language describing at least one assay parameter, such as reagent concentrations or the relative amounts of reagents and sample to be mixed, the duration of the reagent / sample mixture, temperature, buffer conditions, etc.

[0171] composition The present disclosure also provides compositions comprising the modified polymerases described herein. The compositions may include other components in addition to the modified polymerase. For example, the compositions may include a buffer, a nucleotide solution, or a combination thereof. The nucleotide solution may include nucleotides, such as labeled nucleotides, synthetic nucleotides, modified nucleotides, or a combination thereof. In one embodiment, the composition includes a target nucleic acid, such as a library of target nucleic acids. In one embodiment, the composition may include the modified polymerase present with an array, such as a flow cell or beads.

[0172] The present invention is defined in the claims. However, the following provides a non-exhaustive list of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of any other example, embodiment, or aspect described herein.

[0173] Exemplary Embodiments Aspect 1 is a modified archaeal family B DNA polymerase, the modified archaeal family B DNA polymerase comprising an amino acid substitution mutation at a position functionally equivalent to an amino acid in a reference archaeal family B DNA polymerase of SEQ ID NO: 1, the modified archaeal family B The DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO: 1, wherein the amino acid at position Arg58 comprises a mutation to Leu, the amino acid at position Tyr261 comprises a mutation to Gly, the amino acid at position Asn269 comprises a mutation to Gly or Val, the amino acid at position Phe283 comprises a mutation to Ile or Lys, the amino acid at position Pro328 comprises a mutation to Asp, the amino acid at position Met329 comprises a mutation to Thr, the amino acid at position Gln332 comprises a mutation to Ser, the amino acid at position Leu333 comprises a mutation to His or Ile, and the amino acid at position Ser347 comprises Asp, Arg, Glu, or Val, or or Thr, the amino acid at position Asn399 is mutated to Met or Ala, the amino acid at position Phe405 is mutated to Met, Lys or Gln, the amino acid at position Arg406 is mutated to Met, the amino acid at position Ile410 is mutated to Val, the amino acid at position Ile412 is mutated to Val, the amino acid at position Glu458 is mutated to Gly, the amino acid at position the amino acid at position 459 Glu is mutated to Thr or Asp, the amino acid at position 461 Gln is mutated to Trp, the amino acid at position 469 Ala is mutated to Glu, the amino acid at position 481 Tyr is mutated to Ile, the amino acid at position 485 Val is mutated to Asn or Gly or Met or Gln or Ser or Thr, and the amino acid at position 486 Ile is mutated to Leu,the amino acid at position Trp504 is mutated to Asn, the amino acid at position Lys507 is mutated to His or Pro, the amino acid at position Glu511 is mutated to Lys, Met, or Arg, the amino acid at position Trp516 is mutated to He, or Lys, or Met, or Gln, or Leu, the amino acid at position Tyr520 is mutated to Ala, the amino acid at position Ile521 is mutated to Thr, the amino acid at position Met523 is mutated to He or Thr, the amino acid at position Arg526 is mutated to Asn, the amino acid at position Glu527 is mutated to He, and the amino acid at position Leu528, the amino acid is mutated to Thr; at position Ile567, the amino acid is mutated to Leu; at position Asn568, the amino acid is mutated to Gln; at position Leu571, the amino acid is mutated to Phe, Ile, Met, or Trp; at position Glu576, the amino acid is mutated to Leu; at position Glu580, the amino acid is mutated to Ile, Lys, Gln, Arg, Val, or Met; at position Thr590, the amino acid is mutated to Lys; at position Ala595, the amino acid is mutated to Met; at position Ile603, the amino acid is mutated to Asp; or any combination thereof.

[0174] Embodiment 2 is the modified archaeal family B DNA polymerase of embodiment 1, wherein the amino acid at position Leu333 comprises a mutation to His; the amino acid at position Ser347 comprises a mutation to Asp or Arg; the amino acid at position Asn399 comprises a mutation to Met; the amino acid at position Phe405 comprises a mutation to Met; the amino acid at position Tyr481 comprises a mutation to He; the amino acid at position Val485 comprises a mutation to Asn; the amino acid at position Ile486 comprises a mutation to Lys; the amino acid at position Trp504 comprises a mutation to Asn; the amino acid at position Glu511 comprises a mutation to Lys or Met; the amino acid at position Asn568 comprises a mutation to Gln; or the amino acid at position Glu580 comprises a mutation to He, Lys, Gln, Arg, or Val, or any combination thereof.

[0175] Aspect 3 is a modified archaeal family B DNA polymerase according to Aspect 1 or 2, wherein the amino acid at position Asn269 comprises a mutation to Gly or Val, the amino acid at position Leu333 comprises a mutation to Ile, the amino acid at position Ser347 comprises a mutation to Glu or Val, the amino acid at position Asn399 comprises a mutation to Ala, the amino acid at position Phe405 comprises a mutation to Lys or Gln, the amino acid at position Ile412 comprises a mutation to Val, the amino acid at position Glu459 comprises a mutation to Thr, and the amino acid at position Gln461 comprises a mutation to Trp, and the amino acid The amino acid at position Val485 is mutated to Gly or Met or Gln or Ser or Thr, the amino acid at position Glu511 is mutated to Arg, the amino acid at position Trp516 is mutated to Ile or Lys or Met or Gln, the amino acid at position Tyr520 is mutated to Ala, the amino acid at position Ile567 is mutated to Leu, the amino acid at position Glu580 is mutated to Met, or any combination thereof.

[0176] Aspect 4 is a modified archaeal family B DNA polymerase comprising an amino acid substitution mutation at a position functionally equivalent to Glu580 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:1.

[0177] Aspect 5 is a modified archaeal family B DNA polymerase according to aspect 4, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to a polar amino acid.

[0178] Aspect 6 is the modified archaeal family B DNA polymerase according to aspect 4 or 5, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys or Arg.

[0179] Aspect 7 is the modified archaeal family B DNA polymerase of any one of Aspects 4 to 6, wherein the polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Phe405 in the reference archaeal family B DNA polymerase.

[0180] Aspect 8 is the modified archaeal family B DNA polymerase according to any one of Aspects 4 to 7, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0181] Aspect 9 is the modified archaeal family B DNA polymerase according to any one of Aspects 4 to 8, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met.

[0182] Aspect 10 is the modified archaeal Family B DNA polymerase of any one of Aspects 4 to 9, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Arg, and further comprises a substitution mutation at a position functionally equivalent to Phe405, which comprises a mutation to Met.

[0183] Aspect 11 is the modified archaeal family B DNA polymerase of any one of Aspects 4 to 10, wherein the polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Val485 in the reference archaeal family B DNA polymerase.

[0184] Aspect 12 is the modified archaeal family B DNA polymerase of any one of Aspects 4 to 11, wherein the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to a polar amino acid or an uncharged amino acid.

[0185] Aspect 13 is the modified archaeal family B DNA polymerase according to any one of Aspects 4 to 12, wherein the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to Thr.

[0186] Aspect 14 is the modified archaeal Family B DNA polymerase of any one of Aspects 4 to 13, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Val485, which comprises a mutation to Thr.

[0187] Aspect 15 is the modified archaeal family B DNA polymerase of any one of Aspects 4 to 14, wherein the polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Trp516 in the reference archaeal family B DNA polymerase.

[0188] Aspect 16 is the modified archaeal family B DNA polymerase according to any one of Aspects 4 to 15, wherein the substitution mutation at the position functionally equivalent to Trp516 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0189] Aspect 17 is the modified archaeal family B DNA polymerase according to any one of Aspects 4 to 16, wherein the substitution mutation at the position functionally equivalent to Trp516 comprises a mutation to Met or Leu.

[0190] Aspect 18 is the modified archaeal family B DNA polymerase of any one of Aspects 4 to 17, wherein the polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Leu571 in the reference archaeal family B DNA polymerase.

[0191] Aspect 19 is the modified archaeal family B DNA polymerase according to any one of Aspects 4 to 18, wherein the substitution mutation at the position functionally equivalent to Leu571 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0192] Aspect 20 is the modified archaeal family B DNA polymerase of any one of Aspects 4 to 19, wherein the substitution mutation at the position functionally equivalent to Leu571 comprises a mutation to Phe or Met.

[0193] Aspect 21 is the modified archaeal Family B DNA polymerase of any one of Aspects 4 to 20, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys and further comprises a substitution mutation at a position functionally equivalent to Leu571 which comprises a mutation to Phe, or wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Gln and further comprises a substitution mutation at a position functionally equivalent to Leu571 which comprises a mutation to Met.

[0194] Embodiment 22 is the modified archaeal family B DNA polymerase of any one of Embodiments 4 to 21, wherein the polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Ala408, Ile410, or both Ala408 and Ile410 in the reference archaeal family B DNA polymerase.

[0195] Aspect 23 is a modified archaeal family B DNA polymerase according to any one of Aspects 4 to 22, wherein the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to a polar amino acid or an uncharged amino acid.

[0196] Aspect 24 is the modified archaeal family B DNA polymerase according to any one of Aspects 4 to 23, wherein the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to Ser.

[0197] Aspect 25 is the modified archaeal family B DNA polymerase of any one of Aspects 4 to 24, wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0198] Embodiment 26 is the modified archaeal family B DNA polymerase according to any one of embodiments 4 to 25, wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Val.

[0199] Aspect 27 is the modified archaeal Family B DNA polymerase of any one of Aspects 4 to 26, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Trp516, comprising a mutation to Met or Leu.

[0200] Aspect 28 is the modified archaeal Family B DNA polymerase of any one of Aspects 4 to 27, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Ala408 comprises a mutation to Ser, and a substitution mutation at a position functionally equivalent to Ile410 comprises a mutation to Val.

[0201] Aspect 29 is the modified archaeal Family B DNA polymerase of any one of Aspects 4 to 28, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Ala408, which comprises a mutation to Ser.

[0202] Aspect 30 is the modified archaeal Family B DNA polymerase of any one of Aspects 4 to 29, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Phe405, which comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0203] Aspect 31 is a modified archaeal family B DNA polymerase according to any one of Aspects 4 to 30, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met.

[0204] Aspect 32 is the modified archaeal Family B DNA polymerase of any one of Aspects 4 to 31, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Phe405, which comprises a mutation to Met.

[0205] Aspect 33 is the modified archaeal Family B DNA polymerase of any one of Aspects 4 to 32, further comprising a substitution mutation at a position functionally equivalent to Ile410, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid.

[0206] Embodiment 34 is a modified archaeal family B DNA polymerase according to any one of embodiments 4 to 33, wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Val.

[0207] Embodiment 35 is the modified archaeal Family B DNA polymerase of any one of Embodiments 4 to 34, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Ile410, which comprises a mutation to Val.

[0208] Embodiment 36 is a modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Phe405 and Val485 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:1.

[0209] Embodiment 37 is a modified archaeal family B DNA polymerase according to embodiment 36, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0210] Embodiment 38 is a modified archaeal family B DNA polymerase according to embodiment 36 or 37, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met.

[0211] Aspect 39 is a modified archaeal family B DNA polymerase according to any one of Aspects 36 to 38, wherein the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to a non-polar or hydrophobic or uncharged amino acid.

[0212] Embodiment 40 is the modified archaeal family B DNA polymerase of any one of Embodiments 36 to 39, wherein the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to Gly.

[0213] Embodiment 41 is the modified archaeal Family B DNA polymerase of any one of Embodiments 36 to 40, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met and the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to Gly.

[0214] Embodiment 42 is the modified archaeal family B DNA polymerase of any one of Embodiments 36 to 41, wherein the polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Ala408, Ile410, or both Ala408 and Ile410 in the reference archaeal family B DNA polymerase.

[0215] Aspect 43 is a modified archaeal family B DNA polymerase according to any one of Aspects 36 to 42, wherein the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to a polar amino acid or an uncharged amino acid.

[0216] Embodiment 44 is a modified archaeal family B DNA polymerase according to any one of Embodiments 36 to 43, wherein the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to Ser.

[0217] Aspect 45 is a modified archaeal family B DNA polymerase according to any one of Aspects 36 to 44, wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0218] Embodiment 46 is a modified archaeal family B DNA polymerase according to any one of Embodiments 36 to 45, wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Val.

[0219] Aspect 47 is a modified archaeal Family B DNA polymerase according to any one of Aspects 36 to 46, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met, the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to Gly, and further comprises a substitution mutation at a position functionally equivalent to Ala408 which comprises a mutation to Ser, and a substitution mutation at a position functionally equivalent to Ile410 which comprises a mutation to Val.

[0220] Aspect 48 is a modified archaeal Family B DNA polymerase according to any one of Aspects 36 to 47, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met, the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to Gly, and further comprises a substitution mutation at a position functionally equivalent to Ala408 which comprises a mutation to Ser.

[0221] Embodiment 49 is a modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Phe405 and Ile410 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:1.

[0222] Embodiment 50 is a modified archaeal family B DNA polymerase according to embodiment 49, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0223] Embodiment 51 is a modified archaeal family B DNA polymerase according to embodiment 49 or 50, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met.

[0224] Embodiment 52 is a modified archaeal family B DNA polymerase according to any one of embodiments 49 to 51, wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid.

[0225] Embodiment 53 is a modified archaeal family B DNA polymerase according to any one of embodiments 49 to 52, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to Val.

[0226] Embodiment 54 is the modified archaeal Family B DNA polymerase of any one of Embodiments 49 to 53, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met, and the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Val.

[0227] Embodiment 55 is a modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Phe140 and Ser407 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:1.

[0228] Aspect 56 is the modified archaeal family B DNA polymerase of any one of Aspects 49 to 55, wherein the substitution mutation at the position functionally equivalent to Phe140 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0229] Embodiment 57 is a modified archaeal family B DNA polymerase according to any one of embodiments 49 to 56, wherein the substitution mutation at the position functionally equivalent to Phe140 comprises a mutation to Leu.

[0230] Aspect 58 is the modified archaeal family B DNA polymerase of any one of Aspects 49 to 57, wherein the substitution mutation at the position functionally equivalent to Ser407 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0231] Embodiment 59 is the modified archaeal family B DNA polymerase according to any one of embodiments 49 to 58, wherein the substitution mutation at the position functionally equivalent to Ser407 comprises a mutation to Leu.

[0232] Embodiment 60 is the modified archaeal Family B DNA polymerase of any one of Embodiments 49 to 59, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Leu, and the substitution mutation at the position functionally equivalent to Ser407 comprises a mutation to Leu.

[0233] Embodiment 61 is a modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Leu403, Ala408, Ile410, and Gly497 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:1.

[0234] Embodiment 62 is a modified archaeal family B DNA polymerase according to embodiment 61, wherein the substitution mutation at the position functionally equivalent to Leu403 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0235] Embodiment 63 is a modified archaeal family B DNA polymerase according to embodiment 61 or 62, wherein the substitution mutation at the position functionally equivalent to Leu403 comprises a mutation to Met.

[0236] Embodiment 64 is a modified archaeal family B DNA polymerase according to any one of embodiments 61 to 63, wherein the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to a polar amino acid or an uncharged amino acid.

[0237] Embodiment 65 is a modified archaeal family B DNA polymerase according to any one of embodiments 61 to 64, wherein the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to Ser.

[0238] Embodiment 66 is a modified archaeal family B DNA polymerase according to any one of embodiments 61 to 65, wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0239] Embodiment 67 is a modified archaeal family B DNA polymerase according to any one of embodiments 61 to 66, wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Val.

[0240] Embodiment 68 is the modified archaeal family B DNA polymerase of any one of Embodiments 61 to 67, wherein the substitution mutation at the position functionally equivalent to Gly497 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0241] Embodiment 69 is a modified archaeal family B DNA polymerase according to any one of embodiments 61 to 68, wherein the substitution mutation at the position functionally equivalent to Gly497 comprises a mutation to Met.

[0242] Embodiment 70 is a modified archaeal Family B DNA polymerase according to any one of Embodiments 61 to 69, wherein the substitution mutation at the position functionally equivalent to Leu403 comprises a mutation to Met, the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to Ser, the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Val, and the substitution mutation at the position functionally equivalent to Gly497 comprises a mutation to Met.

[0243] Embodiment 71 is a modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Phe405, Ile410, Ile412, Thr514, and Ile521 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:1.

[0244] Embodiment 72 is a modified archaeal family B DNA polymerase according to embodiment 71, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0245] Embodiment 73 is a modified archaeal family B DNA polymerase according to embodiment 71 or 72, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Ile.

[0246] Embodiment 74 is a modified archaeal family B DNA polymerase according to any one of embodiments 71 to 73, wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0247] Embodiment 75 is a modified archaeal family B DNA polymerase according to any one of embodiments 71 to 74, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to Pro.

[0248] Embodiment 76 is a modified archaeal family B DNA polymerase according to any one of embodiments 71 to 75, wherein the substitution mutation at the position functionally equivalent to Ile412 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0249] Embodiment 77 is a modified archaeal family B DNA polymerase according to any one of embodiments 71 to 76, wherein said substitution mutation at said position functionally equivalent to Ile412 comprises a mutation to Met.

[0250] Embodiment 78 is a modified archaeal family B DNA polymerase according to any one of embodiments 71 to 77, wherein the substitution mutation at the position functionally equivalent to Thr514 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0251] Embodiment 79 is a modified archaeal family B DNA polymerase according to any one of embodiments 71 to 78, wherein the substitution mutation at the position functionally equivalent to Thr514 comprises a mutation to Ala.

[0252] Embodiment 80 is a modified archaeal family B DNA polymerase according to any one of embodiments 71 to 79, wherein the substitution mutation at the position functionally equivalent to Ile521 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0253] Embodiment 81 is a modified archaeal family B DNA polymerase according to any one of embodiments 71 to 80, wherein the substitution mutation at the position functionally equivalent to Ile521 comprises a mutation to Ala.

[0254] Aspect 82 is a modified archaeal Family B DNA polymerase according to any of Aspects 71 to 81, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Ile; the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Pro; the substitution mutation at the position functionally equivalent to Ile412 comprises a mutation to Met; the substitution mutation at the position functionally equivalent to Thr514 comprises a mutation to Ala; and the substitution mutation at the position functionally equivalent to Ile521 comprises a mutation to Ala.

[0255] Embodiment 83 is a modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Phe405, Ala408, Ile410, Ile412, Thr514, and Ile521 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) relative to SEQ ID NO:1.

[0256] Embodiment 84 is a modified archaeal family B DNA polymerase according to embodiment 83, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0257] Embodiment 85 is a modified archaeal family B DNA polymerase according to embodiment 83 or 84, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Leu.

[0258] Embodiment 86 is a modified archaeal family B DNA polymerase according to any one of embodiments 83 to 85, wherein the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to a polar amino acid or an uncharged amino acid.

[0259] Embodiment 87 is a modified archaeal family B DNA polymerase according to any one of embodiments 83 to 86, wherein the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to Ser.

[0260] Embodiment 88 is a modified archaeal family B DNA polymerase according to any one of embodiments 83 to 87, wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0261] Embodiment 89 is a modified archaeal family B DNA polymerase according to any one of embodiments 83 to 88, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to Val.

[0262] Embodiment 90 is a modified archaeal family B DNA polymerase according to any one of embodiments 83 to 89, wherein the substitution mutation at the position functionally equivalent to Ile412 comprises a mutation to a polar amino acid or an uncharged amino acid.

[0263] Embodiment 91 is a modified archaeal family B DNA polymerase according to any one of embodiments 83 to 90, wherein said substitution mutation at said position functionally equivalent to Ile412 comprises a mutation to Thr.

[0264] Embodiment 92 is the modified archaeal family B DNA polymerase of any one of embodiments 83 to 91, wherein the substitution mutation at the position functionally equivalent to Thr514 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0265] Embodiment 93 is a modified archaeal family B DNA polymerase according to any one of embodiments 83 to 92, wherein the substitution mutation at the position functionally equivalent to Thr514 comprises a mutation to Ala.

[0266] Embodiment 94 is a modified archaeal family B DNA polymerase according to any one of embodiments 83 to 93, wherein the substitution mutation at the position functionally equivalent to Ile521 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

[0267] Embodiment 95 is a modified archaeal family B DNA polymerase according to any one of embodiments 83 to 94, wherein said substitution mutation at said position functionally equivalent to Ile521 comprises a mutation to Ala.

[0268] Embodiment 96 is the modified archaeal Family B DNA polymerase of any one of Embodiments 83 to 95, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Leu; the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to Ser; the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Val; the substitution mutation at the position functionally equivalent to Ile412 comprises a mutation to Thr; the substitution mutation at the position functionally equivalent to Thr514 comprises a mutation to Ala; and the substitution mutation at the position functionally equivalent to Ile521 comprises a mutation to Ala.

[0269] Aspect 97 is a modified archaeal family B DNA polymerase comprising the amino acid sequence of any of SEQ ID NOs: 2 to 8 or any of SEQ ID NOs: 16 to 32.

[0270] Embodiment 98 is the modified archaeal family B DNA polymerase of any one of embodiments 1 to 97, wherein the polymerase comprises reduced exonuclease activity compared to SEQ ID NO:9.

[0271] Embodiment 99 is a nucleic acid molecule encoding a DNA polymerase as defined in any one of embodiments 1 to 98.

[0272] Embodiment 100 is an expression vector comprising the nucleic acid molecule of embodiment 99.

[0273] Embodiment 101 is a host cell comprising the vector according to embodiment 100.

[0274] Embodiment 102 is a method for incorporating modified nucleotides into a polynucleotide complementary to a target nucleic acid, the method comprising allowing the following components to interact: (i) a modified archaeal family B DNA polymerase of one of embodiments 1-97, (ii) a DNA template, and (iii) a nucleotide solution.

[0275] Example 103 is the method of example 102, wherein the DNA template comprises a clustered array.

[0276] Embodiment 104 is a kit for performing a nucleotide incorporation reaction, comprising the modified archaeal family B DNA polymerase of any of embodiments 1 to 97 and a solution comprising second-generation fully functional nucleotides.

[0277] Embodiment 105 is the kit according to embodiment 104, wherein the second generation fully functional nucleotide comprises a detectable label.

[0278] Embodiment 106 is the kit according to embodiment 104 or 105, wherein the second generation fully functional nucleotide is modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group.

[0279] Embodiment 107 is the kit of any one of embodiments 104 to 106, wherein the second-generation fully functional nucleotide comprises a modified nucleotide molecule comprising a purine or pyrimidine base and a deoxyribose sugar moiety comprising a removable 3'-OH acetal or 3'-OH thiocarbamate blocking group attached to the 3' carbon of the deoxyribose sugar moiety.

[0280] Embodiment 108 is directed to a method wherein the 3′-OH acetal blocking group has the structure:

[0281] [ka] During the ceremony, *** 108. The kit according to any one of embodiments 104 to 107, wherein indicates the point of attachment of the 3'-OH acetal blocking group to the 3' carbon of the modified nucleotide sugar.

[0282] Embodiment 109 is the kit of any one of embodiments 104 to 108, wherein the second generation fully functional nucleotide comprises a nucleotide molecule comprising a purine or pyrimidine base linked to a detectable label via a cleavable linker.

[0283] Embodiment 110 is the kit of any one of embodiments 104 to 109, wherein the detectable label comprises a fluorescent label.

[0284] Embodiment 111 is directed to a method for preparing a cleavable linker comprising:

[0285] [ka] is selected from the group consisting of wherein Z is —O—CH—CH═CH; and n is an integer of 1, 2, 3, 4, or 5; * indicates the point of attachment of the cleavable linker to the purine base or the pyrimidine base; ** indicates the point of attachment of the cleavable linker to the detectable label.

[0286] Embodiment 112 is the kit according to any one of embodiments 104 to 111, further comprising one or more DNA template molecules and / or primers. [Example]

[0287] The present disclosure is illustrated by the following examples, it being understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure described herein.

[0288] Sequencing experiments were used to compare error rates and phasing values. Unless otherwise indicated, experiments were performed on the MiniSeq™ System (Illumina, Inc., San Diego, CA). For example, separate incorporation mixes (IMX) were prepared for each polymerase and used for short reads with either a 30-second or 20-second reaction time. For short reads, the use of a modified kit allowed for six to nine separate incorporation mixes. Modifications to the kit included substituting the deblocking reagent and deblocking scavenger wash buffer. In these experiments, the first and last reads contained internal controls for phasing values ​​and error rate analysis to assess polymerase performance. The first and second reads were used to demonstrate the baseline performance of the control polymerase and custom IMX formulation. The last read was used to assess potential quality degradation throughout the duration of the run. The control used was SEQ ID NO: 1 when polymerases with a single substitution mutation were compared in a sequencing run, and SEQ ID NO: 2 when polymerases with two or more substitution mutations were compared in a sequencing run.

[0289] Primary screening Sequencing experiments were used to compare error rates and phasing values. Unless otherwise indicated, experiments were performed on the MiniSeq™ System (Illumina, Inc., San Diego, CA). For example, a separate incorporation mix (IMX) was prepared for each polymerase and used for short reads with a 30-second reaction time. A modified MiniSeq Mid Output Reagent Cartridge formulation was used to replace the standard polymerase with the polymerase being tested. The use of the modified kit allowed for six to nine separate incorporation mixes. In these experiments, the first and last reads contained internal controls for analysis of phasing values ​​and error rates to assess polymerase performance. The first read was used to demonstrate the baseline performance of the control polymerase and the custom IMX formulation. The last read was used to assess potential quality degradation throughout the duration of the run. Observed phasing values ​​and error rates were reported for each 36-cycle read containing a distinct polymerase. The phasing and error rate values ​​for mutations were normalized to the read 1 control by dividing the observed phasing and error rate by the phasing and error rate of the read 1 control. Mutations with normalized phasing values ​​of 1.2 or less were selected for secondary screening (Figure 2).

[0290] Secondary screening Sequencing experiments were used to compare error rates and phasing values. Unless otherwise indicated, experiments were performed on the MiniSeq™ System (Illumina, Inc., San Diego, CA). For example, a separate integration mix (IMX) was prepared for each polymerase and used in short reads with a 30-second tolerance reaction time. A modified MiniSeq Mid Output Reagent Cartridge formulation was used to replace the standard polymerase with the polymerase being tested. The use of the modified kit allowed for six to nine separate integration mixes. In these experiments, the first, second, and final reads contained internal controls for analysis of phasing values ​​and error rates to evaluate polymerase performance. The first read was used to demonstrate the baseline performance of the control polymerase and custom IMX formulation under permissive conditions with a 30-second tolerance reaction time. The second read was used to demonstrate the performance of the control polymerase and custom IMX formulation under stressed conditions with a 20-second tolerance reaction time. The final read value was used to assess potential quality degradation throughout the duration of the run, also under stress conditions, with a 20-second allowable reaction time. Observed phasing values ​​and error rates were reported for each 36-cycle read containing a distinct polymerase. Phasing and error rate values ​​for mutations were normalized to the read 2 control. Normalized values ​​were obtained by dividing the observed phasing and error rates by those of the read 2 control. Mutations with an average normalized value of 0.5 or less were classified as Tier 1 (Figure 3). Mutations with an average normalized value greater than 0.5 but less than or equal to 1.0 were classified as Tier 2 (Figure 4). Mutations with an average normalized phasing value greater than 1.0 were not classified.

[0291] Tier 1 and Tier 2 mutations were recombined to further identify any further improvements to sequencing performance - some exemplary double mutations are shown in Figure 5.

[0292] Finally, in some examples, these double mutations or similar recombinations may be used as new scaffolds to which further mutations are added prior to validation in a 150-cycle run (Figure 6) - an example of a 151-cycle validation run.

[0293] Effective runs (150 cycles) In some examples, polymerases were prepared for long runs (Figure 7). A modified MiniSeq Mid Output Reagent Cartridge formulation was used to replace the standard polymerase with the polymerase being tested. Incubation times of IMX on the flow cell were varied as described in the Examples herein. Phasing values ​​and error rates were reported and compared to those observed for a control under the same conditions as described.

[0294] The complete disclosures of all patents, patent applications, and publications, as well as electronically available materials cited herein (e.g., nucleotide sequence submissions in GenBank and RefSeq, amino acid sequence submissions in SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq), are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, figures, materials and methods, and / or experimental data) are likewise incorporated by reference in their entirety. In the event of a conflict between the disclosure of this application and the disclosure of a document incorporated by reference herein, the disclosure of this application shall control. The foregoing detailed description and examples are provided for clarity of understanding only. No unnecessary limitations should be understood therefrom. The disclosure is not limited to the exact details shown and described, since variations obvious to those skilled in the art are included in the disclosure defined by the claims.

[0295] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and the like used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the scope of the claims to the doctrine of equivalents, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0296] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviations found in their respective testing measurements.

[0297] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless specifically stated.

Claims

1. a modified archaeal family B DNA polymerase, the modified archaeal family B DNA polymerase comprising an amino acid substitution mutation at a position functionally equivalent to an amino acid in a reference archaeal family B DNA polymerase of SEQ ID NO: 1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO: 1; the amino acid at position Arg58 comprises a mutation to Leu, the amino acid at position Tyr261 comprises a mutation to Gly, the amino acid at position Asn269 comprises a mutation to Gly or Val, the amino acid at position Phe283 comprises a mutation to He or Lys; the amino acid at position Pro328 comprises a mutation to Asp, the amino acid at position 329 of Met comprises a mutation to Thr; the amino acid at position Gln332 comprises a mutation to Ser, the amino acid at position Leu333 comprises a mutation to His or Ile, the amino acid at position Ser347 comprises a mutation to Asp or Arg or Glu or Val or Thr, the amino acid at position Asn399 comprises a mutation to Met or Ala, the amino acid at position Phe405 comprises a mutation to Met or Lys or Gln, the amino acid at position Arg406 comprises a mutation to Met, the amino acid at position Ile410 contains a mutation to Val, the amino acid at position Ile412 comprises a mutation to Val, the amino acid at position Glu458 comprises a mutation to Gly, the amino acid at position Glu459 comprises a mutation to Thr or Asp, the amino acid at position Gln461 comprises a mutation to Trp, the amino acid at position Ala469 comprises a mutation to Glu, the amino acid at position Tyr481 comprises a mutation to He, the amino acid at position Val485 comprises a mutation to Asn, Gly, Met, Gln, Ser, or Thr, the amino acid at position Ile486 contains a mutation to Leu, the amino acid at position Trp504 comprises a mutation to Asn, the amino acid at position Lys507 comprises a mutation to His or Pro, the amino acid at position Glu511 comprises a mutation to Lys, Met, or Arg, the amino acid at position Trp516 comprises a mutation to He or Lys or Met or Gln or Leu; the amino acid at position Tyr520 comprises a mutation to Ala, the amino acid at position Ile521 comprises a mutation to Thr, the amino acid at position Met523 comprises a mutation to He or Thr, the amino acid at position Arg526 comprises a mutation to Asn, the amino acid at position Glu527 comprises a mutation to He, the amino acid at position Leu 528 contains a mutation to Thr, the amino acid at position Ile567 contains a mutation to Leu, the amino acid at position Asn568 comprises a mutation to Gln, the amino acid at Leu position 571 comprises a mutation to Phe or Ile or Met or Trp, the amino acid at position Glu576 comprises a mutation to Leu, the amino acid at position Glu580 comprises a mutation to Ile or Lys or Gln or Arg or Val or Met, the amino acid at position Thr590 comprises a mutation to Lys, the amino acid at position Ala595 comprises a mutation to Met, or A modified archaeal family B DNA polymerase, wherein the amino acid is at position Ile603 and comprises a mutation to Asp.

2. the amino acid at position Leu333 comprises a mutation to His, the amino acid at position Ser347 comprises a mutation to Asp or Arg, the amino acid at position Asn399 comprises a mutation to Met, the amino acid at position Phe405 comprises a mutation to Met, the amino acid at position Tyr481 comprises a mutation to He, the amino acid at position Val485 comprises a mutation to Asn, the amino acid at position Ile486 comprises a mutation to Lys, the amino acid at position Trp504 comprises a mutation to Asn, the amino acid at position Glu511 comprises a mutation to Lys or Met, the amino acid at position Asn568 comprises a mutation to Gln, or 2. The modified archaeal Family B DNA polymerase of claim 1, wherein the amino acid at position Glu580 comprises a mutation to Ile or Lys or Gln or Arg or Val.

3. the amino acid at position Asn269 comprises a mutation to Gly or Val, the amino acid at position Leu333 comprises a mutation to He, the amino acid at position Ser347 comprises a mutation to Glu or Val, the amino acid at position Asn399 comprises a mutation to Ala, the amino acid at position Phe405 comprises a mutation to Lys or Gln, the amino acid at position Ile412 comprises a mutation to Val, the amino acid at position Glu459 comprises a mutation to Thr, the amino acid at position Gln461 comprises a mutation to Trp, the amino acid at position Val485 comprises a mutation to Gly or Met or Gln or Ser or Thr, the amino acid at position Glu511 comprises a mutation to Arg, the amino acid at position Trp516 comprises a mutation to He or Lys or Met or Gln, the amino acid at position Tyr520 comprises a mutation to Ala, the amino acid at position Ile567 comprises a mutation to Leu, or 2. The modified archaeal Family B DNA polymerase of claim 1, wherein the amino acid is at position Glu580 and comprises a mutation to Met.

4. 1. A modified archaeal family B DNA polymerase comprising an amino acid substitution mutation at a position functionally equivalent to Glu580 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:

1.

5. 5. The modified archaeal Family B DNA polymerase of claim 4, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to a polar amino acid.

6. 6. The modified archaeal Family B DNA polymerase of claim 5, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys or Arg.

7. 5. The modified archaeal Family B DNA polymerase of claim 4, wherein said polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Phe405 in said reference archaeal Family B DNA polymerase.

8. 8. The modified archaeal Family B DNA polymerase of claim 7, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

9. 9. The modified archaeal Family B DNA polymerase of claim 8, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met.

10. 9. The modified archaeal Family B DNA polymerase of claim 8, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Arg, and further comprises a substitution mutation at a position functionally equivalent to Phe405 comprises a mutation to Met.

11. 5. The modified archaeal Family B DNA polymerase of claim 4, wherein said polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Val485 in said reference archaeal Family B DNA polymerase.

12. 12. The modified archaeal Family B DNA polymerase of claim 11, wherein the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to a polar amino acid or an uncharged amino acid.

13. 13. The modified archaeal Family B DNA polymerase of claim 12, wherein the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to Thr.

14. 13. The modified archaeal Family B DNA polymerase of claim 12, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Val485 comprises a mutation to Thr.

15. 5. The modified archaeal Family B DNA polymerase of claim 4, wherein said polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Trp516 in said reference archaeal Family B DNA polymerase.

16. 16. The modified archaeal Family B DNA polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Trp516 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

17. 17. The modified archaeal Family B DNA polymerase of claim 16, wherein the substitution mutation at the position functionally equivalent to Trp516 comprises a mutation to Met or Leu.

18. 5. The modified archaeal Family B DNA polymerase of claim 4, wherein said polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Leu571 in said reference archaeal Family B DNA polymerase.

19. 19. The modified archaeal Family B DNA polymerase of claim 18, wherein the substitution mutation at the position functionally equivalent to Leu571 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

20. 20. The modified archaeal Family B DNA polymerase of claim 19, wherein the substitution mutation at the position functionally equivalent to Leu571 comprises a mutation to Phe or Met.

21. 20. The modified archaeal Family B DNA polymerase of claim 19, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys and further comprises a substitution mutation at a position functionally equivalent to Leu571 that comprises a mutation to Phe, or wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Gln and further comprises a substitution mutation at a position functionally equivalent to Leu571 that comprises a mutation to Met.

22. 5. The modified archaeal Family B DNA polymerase of claim 4, wherein said polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Ala408, Ile410, or both Ala408 and Ile410 in said reference archaeal Family B DNA polymerase.

23. 23. The modified archaeal Family B DNA polymerase of claim 22, wherein the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to a polar amino acid or an uncharged amino acid.

24. 24. The modified archaeal Family B DNA polymerase of claim 23, wherein said substitution mutation at said position functionally equivalent to Ala408 comprises a mutation to Ser.

25. 23. The modified archaeal Family B DNA polymerase of claim 22, wherein the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

26. 26. The modified archaeal Family B DNA polymerase of claim 25, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to Val.

27. 5. The modified archaeal Family B DNA polymerase of claim 4, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Trp516, comprising a mutation to Met or Leu.

28. 5. The modified archaeal Family B DNA polymerase of claim 4, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Ala408 comprising a mutation to Ser, and a substitution mutation at a position functionally equivalent to Ile410 comprising a mutation to Val.

29. 5. The modified archaeal Family B DNA polymerase of claim 4, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Ala408 comprises a mutation to Ser.

30. 5. The modified archaeal Family B DNA polymerase of claim 4, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Phe405 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

31. 31. The modified archaeal Family B DNA polymerase of claim 30, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met.

32. 32. The modified archaeal Family B DNA polymerase of claim 31 , wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Phe405 comprises a mutation to Met.

33. 5. The modified archaeal Family B DNA polymerase of claim 4, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Ile410, comprising a mutation to a non-polar amino acid.

34. 34. The modified archaeal Family B DNA polymerase of claim 33, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to Val.

35. 35. The modified archaeal Family B DNA polymerase of claim 34, wherein the substitution mutation at the position functionally equivalent to Glu580 comprises a mutation to Lys, and further comprises a substitution mutation at a position functionally equivalent to Ile410 comprises a mutation to Val.

36. 1. A modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Phe405 and Val485 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:

1.

37. 37. The modified archaeal Family B DNA polymerase of claim 36, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

38. 38. The modified archaeal Family B DNA polymerase of claim 37, wherein said substitution mutation at said position functionally equivalent to Phe405 comprises a mutation to Met.

39. 37. The modified archaeal Family B DNA polymerase of claim 36, wherein said substitution mutation at said position functionally equivalent to Val485 comprises a mutation to a non-polar amino acid, or a hydrophobic amino acid, or an uncharged amino acid.

40. 38. The modified archaeal Family B DNA polymerase of claim 37, wherein said substitution mutation at said position functionally equivalent to Val485 comprises a mutation to Gly.

41. 37. The modified archaeal Family B DNA polymerase of claim 36, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met and the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to Gly.

42. 37. The modified archaeal Family B DNA polymerase of claim 36, wherein said polymerase further comprises an amino acid substitution mutation at a position functionally equivalent to Ala408, Ile410, or both Ala408 and Ile410 in said reference archaeal Family B DNA polymerase.

43. 43. The modified archaeal Family B DNA polymerase of claim 42, wherein said substitution mutation at said position functionally equivalent to Ala408 comprises a mutation to a polar amino acid or an uncharged amino acid.

44. 44. The modified archaeal Family B DNA polymerase of claim 43, wherein said substitution mutation at said position functionally equivalent to Ala408 comprises a mutation to Ser.

45. 43. The modified archaeal Family B DNA polymerase of claim 42, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

46. 46. ​​The modified archaeal Family B DNA polymerase of claim 45, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to Val.

47. 37. The modified archaeal Family B DNA polymerase of claim 36, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met, the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to Gly, and further comprises a substitution mutation at a position functionally equivalent to Ala408 comprising a mutation to Ser and a substitution mutation at a position functionally equivalent to Ile410 comprising a mutation to Val.

48. 37. The modified archaeal Family B DNA polymerase of claim 36, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met, the substitution mutation at the position functionally equivalent to Val485 comprises a mutation to Gly, and further comprises a substitution mutation at a position functionally equivalent to Ala408 comprises a mutation to Ser.

49. 1. A modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Phe405 and Ile410 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:

1.

50. 50. The modified archaeal Family B DNA polymerase of claim 49, wherein said substitution mutation at said position functionally equivalent to Phe405 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

51. 51. The modified archaeal Family B DNA polymerase of claim 50, wherein said substitution mutation at said position functionally equivalent to Phe405 comprises a mutation to Met.

52. 50. The modified archaeal Family B DNA polymerase of claim 49, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid.

53. 53. The modified archaeal Family B DNA polymerase of claim 52, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to Val.

54. 50. The modified archaeal Family B DNA polymerase of claim 49, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Met and the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Val.

55. 1. A modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Phe140 and Ser407 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:

1.

56. 56. The modified archaeal Family B DNA polymerase of claim 55, wherein said substitution mutation at said position functionally equivalent to Phe140 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

57. 57. The modified archaeal Family B DNA polymerase of claim 56, wherein said substitution mutation at said position functionally equivalent to Phe140 comprises a mutation to Leu.

58. 56. The modified archaeal Family B DNA polymerase of claim 55, wherein said substitution mutation at said position functionally equivalent to Ser407 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

59. 59. The modified archaeal Family B DNA polymerase of claim 58, wherein said substitution mutation at said position functionally equivalent to Ser407 comprises a mutation to Leu.

60. 56. The modified archaeal Family B DNA polymerase of claim 55, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Leu and the substitution mutation at the position functionally equivalent to Ser407 comprises a mutation to Leu.

61. 1. A modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Leu403, Ala408, Ile410, and Gly497 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:

1.

62. 62. The modified archaeal Family B DNA polymerase of claim 61, wherein said substitution mutation at said position functionally equivalent to Leu403 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

63. 63. The modified archaeal Family B DNA polymerase of claim 62, wherein said substitution mutation at said position functionally equivalent to Leu403 comprises a mutation to Met.

64. 62. The modified archaeal Family B DNA polymerase of claim 61, wherein said substitution mutation at said position functionally equivalent to Ala408 comprises a mutation to a polar amino acid or an uncharged amino acid.

65. 65. The modified archaeal Family B DNA polymerase of claim 64, wherein said substitution mutation at said position functionally equivalent to Ala408 comprises a mutation to Ser.

66. 62. The modified archaeal Family B DNA polymerase of claim 61, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

67. 67. The modified archaeal Family B DNA polymerase of claim 66, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to Val.

68. 62. The modified archaeal Family B DNA polymerase of claim 61, wherein said substitution mutation at said position functionally equivalent to Gly497 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

69. 69. The modified archaeal Family B DNA polymerase of claim 68, wherein said substitution mutation at said position functionally equivalent to Gly497 comprises a mutation to Met.

70. 62. The modified archaeal Family B DNA polymerase of claim 61 , wherein the substitution mutation at the position functionally equivalent to Leu403 comprises a mutation to Met, the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to Ser, the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Val, and the substitution mutation at the position functionally equivalent to Gly497 comprises a mutation to Met.

71. 1. A modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Phe405, Ile410, Ile412, Thr514, and Ile521 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:

1.

72. 72. The modified archaeal Family B DNA polymerase of claim 71, wherein said substitution mutation at said position functionally equivalent to Phe405 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

73. 73. The modified archaeal Family B DNA polymerase of claim 72, wherein said substitution mutation at said position functionally equivalent to Phe405 comprises a mutation to Leu.

74. 72. The modified archaeal Family B DNA polymerase of claim 71, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

75. 75. The modified archaeal Family B DNA polymerase of claim 74, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to Pro.

76. 72. The modified archaeal Family B DNA polymerase of claim 71, wherein said substitution mutation at said position functionally equivalent to Ile412 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

77. 77. The modified archaeal Family B DNA polymerase of claim 76, wherein said substitution mutation at said position functionally equivalent to Ile412 comprises a mutation to Met.

78. 72. The modified archaeal Family B DNA polymerase of claim 71, wherein said substitution mutation at said position functionally equivalent to Thr514 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

79. 79. The modified archaeal Family B DNA polymerase of claim 78, wherein said substitution mutation at said position functionally equivalent to Thr514 comprises a mutation to Ala.

80. 72. The modified archaeal Family B DNA polymerase of claim 71, wherein said substitution mutation at said position functionally equivalent to Ile521 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

81. 81. The modified archaeal Family B DNA polymerase of claim 80, wherein said substitution mutation at said position functionally equivalent to Ile521 comprises a mutation to Ala.

82. 72. The modified archaeal Family B DNA polymerase of Claim 71, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Ile, the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Pro, the substitution mutation at the position functionally equivalent to Ile412 comprises a mutation to Met, the substitution mutation at the position functionally equivalent to Thr514 comprises a mutation to Ala, and the substitution mutation at the position functionally equivalent to Ile521 comprises a mutation to Ala.

83. 1. A modified archaeal family B DNA polymerase comprising amino acid substitution mutations at positions functionally equivalent to Phe405, Ala408, Ile410, Ile412, Thr514, and Ile521 in a reference archaeal family B DNA polymerase of SEQ ID NO:1, wherein the modified archaeal family B DNA polymerase is capable of incorporating modified nucleotides comprising a 3'-OH acetal blocking group or a 3'-OH thiocarbamate blocking group with (i) a lower error rate, (ii) a lower phasing rate, or both (i) and (ii) compared to SEQ ID NO:

1.

84. 84. The modified archaeal Family B DNA polymerase of claim 83, wherein said substitution mutation at said position functionally equivalent to Phe405 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

85. 85. The modified archaeal Family B DNA polymerase of claim 84, wherein said substitution mutation at said position functionally equivalent to Phe405 comprises a mutation to Leu.

86. 84. The modified archaeal Family B DNA polymerase of claim 83, wherein said substitution mutation at said position functionally equivalent to Ala408 comprises a mutation to a polar amino acid or an uncharged amino acid.

87. 87. The modified archaeal Family B DNA polymerase of claim 86, wherein said substitution mutation at said position functionally equivalent to Ala408 comprises a mutation to Ser.

88. 84. The modified archaeal Family B DNA polymerase of claim 83, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

89. 89. The modified archaeal Family B DNA polymerase of claim 88, wherein said substitution mutation at said position functionally equivalent to Ile410 comprises a mutation to Val.

90. 84. The modified archaeal Family B DNA polymerase of claim 83, wherein said substitution mutation at said position functionally equivalent to Ile412 comprises a mutation to a polar amino acid or an uncharged amino acid.

91. 91. The modified archaeal Family B DNA polymerase of claim 90, wherein said substitution mutation at said position functionally equivalent to Ile412 comprises a mutation to Thr.

92. 84. The modified archaeal Family B DNA polymerase of claim 83, wherein said substitution mutation at said position functionally equivalent to Thr514 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

93. 93. The modified archaeal Family B DNA polymerase of claim 92, wherein said substitution mutation at said position functionally equivalent to Thr514 comprises a mutation to Ala.

94. 84. The modified archaeal Family B DNA polymerase of claim 83, wherein said substitution mutation at said position functionally equivalent to Ile521 comprises a mutation to a non-polar amino acid or a hydrophobic amino acid.

95. 95. The modified archaeal Family B DNA polymerase of claim 94, wherein said substitution mutation at said position functionally equivalent to Ile521 comprises a mutation to Ala.

96. 84. The modified archaeal Family B DNA polymerase of Claim 83, wherein the substitution mutation at the position functionally equivalent to Phe405 comprises a mutation to Leu, the substitution mutation at the position functionally equivalent to Ala408 comprises a mutation to Ser, the substitution mutation at the position functionally equivalent to Ile410 comprises a mutation to Val, the substitution mutation at the position functionally equivalent to Ile412 comprises a mutation to Thr, the substitution mutation at the position functionally equivalent to Thr514 comprises a mutation to Ala, and the substitution mutation at the position functionally equivalent to Ile521 comprises a mutation to Ala.

97. A modified archaeal family B DNA polymerase comprising the amino acid sequence of any one of SEQ ID NOs:2 to 8, or any one of SEQ ID NOs:16 to 32.

98. 98. The modified archaeal Family B DNA polymerase of claim 1, 4, 36, 49, 55, 61, 71, 83, or 97, wherein the polymerase comprises reduced exonuclease activity compared to SEQ ID NO:

9.

99. A nucleic acid molecule encoding a DNA polymerase as defined in any one of claims 1 to 98.

100. 100. An expression vector comprising the nucleic acid molecule of claim 99.

101. A host cell comprising the vector of claim 100.

102. 10. A method for incorporating modified nucleotides into a polynucleotide complementary to a target nucleic acid, comprising allowing the following components to interact: (i) a modified archaeal family B DNA polymerase of claim 1, 4, 36, 49, 55, 61, 71, 83, or 97; (ii) a DNA template; and (iii) a nucleotide solution.

103. 103. The method of claim 102, wherein the DNA template comprises a clustered array.

104. 103. A kit for performing a nucleotide incorporation reaction, comprising a solution comprising the modified archaeal Family B DNA polymerase of claim 1, 4, 36, 49, 55, 61, 71, 83, or 97 and second-generation fully functional nucleotides.

105. 105. The kit of claim 104, wherein the second generation fully functional nucleotide comprises a detectable label.

106. 105. The kit of claim 104, wherein said second generation fully functional nucleotides are modified at the 3' sugar hydroxyl such that said substituent is larger in size than the naturally occurring 3' hydroxyl group.

107. 107. The kit of claim 106, wherein the second-generation fully functional nucleotide comprises a modified nucleotide molecule comprising a purine or pyrimidine base and a deoxyribose sugar moiety comprising a removable 3'-OH acetal or 3'-OH thiocarbamate blocking group attached to the 3' carbon of the deoxyribose sugar moiety.

108. the 3'-OH acetal blocking group has the structure: 【Chemical 1】 During the ceremony, ＊＊＊ 108. The kit of claim 107, wherein indicates the point of attachment of the 3'-OH acetal blocking group to the 3' carbon of the modified nucleotide sugar.

109. 105. The kit of claim 104, wherein the second generation fully functional nucleotide comprises a nucleotide molecule comprising a purine or pyrimidine base linked to a detectable label via a cleavable linker.

110. 110. The kit of claim 109, wherein the detectable label comprises a fluorescent label.

111. the cleavable linker 【Chemistry 2】 is selected from the group consisting of In the formula, Z is —O—CH ２ -CH=CH ２ and n is an integer of 1, 2, 3, 4, or 5; ＊ indicates the point of attachment of the cleavable linker to the purine base or the pyrimidine base; ＊＊ 110. The kit of claim 109, wherein: indicates the point of attachment of the cleavable linker to the detectable label.

112. 105. The kit of claim 104, further comprising one or more DNA template molecules and / or primers.

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