RNase H2 mutants that reduce primer dimerization and off-target amplification in rhPCR-based amplification product sequencing using high-fidelity DNA polymerases.

Abstract

The present invention relates to a hybrid RNase H2 protein comprising fragments of amino acid sequences derived from the organisms Pyrococcus abyssi (Pa), Thermocox kodakarensis (T. kod) and Pyrococcus furiosus, as well as methods of using same to improve mismatch discrimination and activity in high fidelity DNA polymerase buffers.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 130,548, filed on 24 December 2020 and titled "RNASE H2 MUTANTS THAT ENHANCE MISMATCH DISCRIMINATION AND ACTIVITY IN HIGH-FIDELITY POLYMERASE BUFFER," and U.S. Provisional Patent Application No. 63 / 277,273, filed on 9 November 2021 and titled "RNASE H2 MUTANTS THAT REDUCE PRIMER DIMERS AND OFF-TARGET AMPLIFICATION IN RHPCR-BASED AMPLICON SEQUENCING WITH HIGH-FIDELITY DNA POLYMERASES," whose contents are incorporated herein by reference in their entirety.

[0002] Field of the present invention The present invention relates to a hybrid enzyme variant of type II RNase H (hereinafter referred to as RNase H2), and to a method for cleaving nucleic acid strands to initiate, assist, monitor, or carry out a biological assay. [Background technology]

[0003] The family of RNase H2 enzymes is broadly characterized. These enzymes possess substrate specificity for cleaving single ribonucleotides embedded within DNA sequences (in double-stranded form) (Eder et al., (1993), Biochimie, 75, 123-126). Interestingly, the cleavage occurs at the 5' end of the RNA residue (see Scheme I). An overview of these enzymes, their properties, and applications for biological assays is summarized in Walder et al., U.S. Patent No. 8,911,948B2.

[0004] [ka]

[0005] The RNase H2 enzyme isolated from the hyperthermophilic bacterium Pyrococcus abyssi (Pa) cleaves all other ribonucleotides embedded within nucleic acid chains made from DNA at the 5' end. RNase H2-dependent PCR (rhPCR) improves PCR specificity by using the thermostable RNase H2 enzyme to remove a blocking group at the 3' end and cleave primers containing a single ribonucleotide near the 3' end (Dobosy et al., 2011; U.S. Patent No. 8,911,948B2). This primer is initially unable to be extended by DNA polymerase, but becomes extendable after the blocking group is removed from the 3' end by RNase H2. PaRNase H2 is sensitive to single-nucleotide mismatches near ribonucleotides within the DNA-RNA heteroduplex and cleaves mismatch-containing templates at a very low rate. This allows for the preferential cleavage and extension of perfectly matched double helices. This results in increased specificity in rhPCR reactions and reduced primer dimerization and other off-target amplification.

[0006] Despite the enhancement it brings to PCR specificity, rhPCR currently has limitations. The apparent mismatch recognition by rhPCR is lower than the theoretically achievable level. WT PaRNase H2 highly recognizes single-base mismatches directly opposite RNA bases, but its efficiency varies depending on the nature of the mismatch. In addition, native enzymes are also relatively limited in recognizing mismatches at the immediate 5' or 3' end of RNA bases. Despite these limitations, the placement of mutations at these locations can be advantageous. For example, a mismatch at the immediate 5' end of RNA can be used as a secondary selection step in PCR amplification after primer cleavage, involving the use of a discriminative DNA polymerase (such as H784Q Thermus Aquatics DNA Polymerase) (see U.S. Patent Application No. 15 / 361280). Since mismatch recognition occurs not just once during template transition, but per cycle, the placement of a mismatch at the 3' end of RNA reduces the likelihood of template transition.

[0007] Despite the usefulness of rhPCR, mismatch recognition by wild-type PaRNase H2 results in some amplification of primer dimers, which can be a "missing" consequence. Primer dimers generated during sequencing library amplification bind to the Illumina flow cell and undergo sequencing, but are problematic because they do not yield meaningful data. High levels of primer dimers reduce the read fraction mapped to the target of interest, ultimately reducing assay sensitivity or requiring a significant increase in sequencing costs to generate the number of on-target reads necessary for detecting low-frequency variants. Detection of low-frequency variants can also be affected by the introduction of amplification errors during PCR. Error rates can be reduced by using high-fidelity DNA polymerases and optimized buffer conditions. However, these buffer conditions reduce the enzymatic activity and mismatch sensitivity of wild-type PaRNase H2 in rhPCR.

[0008] It has already been shown that the use of RNase H2 mutants, created by partial recombination of the amino acid sequence of wild-type PaRNase H2 using sequences derived from other related molecular species, results in improved RNase H2 enzyme activity in high-fidelity DNA polymerase buffer. Two of these mutants, Q48R SEL29 RNase H2 and Al07V SEL29 RNase H2, have been shown to exhibit improved enzyme activity when using Thermococcus kodakarensis (KOD) DNA polymerase reaction buffer. In addition, both mutants show enhanced recognition of mismatches at the opposite end of RNA bases, as well as at the 3' and 5' ends of RNA bases, compared to wild-type PaRNase H2. See U.S. Provisional Patent Application No. 63 / 130,548, filed on 24 December 2020, titled "RNASE H2 MUTANTS THAT ENHANCE MISMATCH DISCRIMINATION AND ACTIVITY IN HIGH-FIDELITY POLYMERASE BUFFER" (Agent Reference Number: IDT01-018-PRO), which is incorporated herein by reference in its entirety. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] U.S. Patent No. 8911948B2 [Patent Document 2] U.S. Patent No. 8911948B2 [Patent Document 3] U.S. Patent Application No. 15 / 361280 [Patent Document 4] U.S. Patent Provisional Application No. 63 / 130548 [Non-patent literature]

[0010] [Non-Patent Document 1] Eder, et al., (1993), Biochimie, 75, 123-126

Summary of the Invention

Problems to be Solved by the Invention

[0011] The present disclosure relates to Q48R SEL29 RNase H2, one of these novel hybrid RNase H2 enzyme mutants, in a multiplex rhAmpSeq workflow containing a high fidelity DNA polymerase and buffer. Compared to wild-type P.a. RNase H2, Q48R SEL29 RNase H2 reduces primer dimers generated during PCR amplification, thereby improving the mapping rate and on-target rate. While improving these metrics, Q48R SEL29 RNase H2 had no effect on other highly important sequencing metrics, including the uniformity of amplification products, the dropout rate of amplification products, and the uniform distribution of amplification products.

Means for Solving the Problems

[0012] (Summary of the Invention) In a first aspect, a hybrid RNase H2 protein is provided. The hybrid RNase H2 protein comprises a fragment of an amino acid sequence derived from the organisms Pyrococcus abyssi (P.a.), Thermococcus kodakaraensis (T.kod) and Pyrococcus furiosus.

[0013] In a second aspect, a recombinant nucleic acid encoding any of the hybrid RNase H2 proteins disclosed herein is provided.

[0014] In a third embodiment, a method for performing primer extension is provided. The method comprises the step of contacting a hybrid RNase H2 protein, as disclosed herein, with a primer, a polynucleotide template, a nucleoside triphosphate, and a DNA polymerase, under conditions suitable for a primer extension method, thereby producing an extension primer.

[0015] In a fourth embodiment, a reaction mixture is provided. The reaction mixture comprises a hybrid RNase H2 protein, at least one primer, a polynucleotide template, a nucleoside triphosphate, and a DNA polymerase, as described herein.

[0016] In a fifth embodiment, a method for performing rhPCR is provided. The method includes the step of performing primer extension using a hybrid RNase H2, DNA polymerase, and primers as described herein.

[0017] A sixth embodiment provides a method for amplifying a target DNA sequence. The method comprises several steps. The first step is to prepare a reaction mixture comprising: (i) an oligonucleotide primer having a cleavage domain cleavable by an RNase H2 enzyme, located at the 5' end of a blocking group linked at or near the 3' end of the oligonucleotide primer, wherein the blocking group prevents primer elongation and / or inhibits the use of the oligonucleotide primer as a template for DNA synthesis; (ii) a sample nucleic acid, which may or may not be a target sequence; (iii) a DNA polymerase; and (iv) a hybrid RNase H2 protein disclosed herein. The second step comprises hybridizing the oligonucleotide primer with the target DNA sequence to form a double-stranded substrate. The third step is to remove the blocking group from the oligonucleotide primer by cleaving the hybridized oligonucleotide primer at a cleavage site within the cleavage domain or at a cleavage site adjacent to the cleavage domain using a hybrid RNase H2 enzyme.

[0018] In a seventh embodiment, a kit for preparing extension primers is provided. The kit comprises at least one container providing the hybrid RNase H2 protein disclosed herein.

[0019] In an eighth aspect, a kit for performing amplification of a target DNA sequence is provided. The kit comprises a reaction buffer containing RNase H2 as described herein and a high-fidelity archaeal DNA polymerase.

[0020] In a ninth aspect, a method for preparing a library of amplification products of template nucleic acids is provided. The method comprises several steps. A first step is to form a mixture comprising a nucleic acid population, at least one blocked cleavable primer, a hybrid RNase H2 protein, dNTPs, DNA polymerase, and a buffer, such that a hybrid double helix is ​​formed between the nucleic acid population and at least one blocked cleavable primer in the mixture. A second step is to cleave at least one blocked cleavable primer with the hybrid RNase H2 protein to produce at least one active primer that can be extended by DNA polymerase. A third step is to extend at least one active primer with DNA polymerase in a buffer under conditions that allow amplification of one or more template nucleic acids from the nucleic acid population, thereby producing amplification products of the template nucleic acids. In the first related matter, the hybrid RNase H2 protein is selected from Q48R SEL29 (SEQ ID NO: 18) or other hybrid RNase H2 proteins. In the second related matter, the DNA polymerase is KOD DNA polymerase or other high-fidelity archaeal DNA polymerase. In the third related matter, the buffer is high-fidelity archaeal DNA polymerase buffer.

[0021] In a tenth aspect, a method for performing massively parallel sequencing is provided. The method comprises several steps. The first step is to prepare a library population of template nucleic acids using a nucleic acid population, a hybrid RNase H2 mutant protein, at least one blocked cleavable primer, DNA polymerase, dNTPs and buffers in a PCR method. The second step is to sequence several desired template nucleic acids derived from the library population of template nucleic acids. In the first aspect, the hybrid RNase H2 protein is selected from Q48R SEL29 (SEQ ID NO: 18) or other hybrid RNase H2 proteins.

[0022] In an eleventh embodiment, a method is provided for detecting an SNP-containing nucleic acid template from a nucleic acid template amplification product library. The method comprises several steps. A first step is to form a mixture comprising: a nucleic acid template amplification product library; at least one blocked cleavable primer; a hybrid mutant RNase H2 protein; dNTPs; DNA polymerase; and a buffer. A hybrid duplex is formed between at least one blocked cleavable primer in the mixture and an SNP-containing nucleic acid template in the nucleic acid template amplification product library. A second step is to cleave at least one blocked cleavable primer of the hybrid duplex with the hybrid RNase H2 protein to create at least one active primer that can be extended by DNA polymerase on the hybrid duplex. A third step is to extend at least one active primer in the duplex with DNA polymerase in a buffer under conditions that allow amplification of one or more template nucleic acids from the nucleic acid template amplification product library, thereby detecting an SNP-containing nucleic acid template. In the first related matter, the method comprises a hybrid mutant RNase H2 protein selected from Q48R SEL29 (SEQ ID NO: 18) or other hybrid mutant RNase H2 proteins. In the second related matter, the method comprises a buffer which is a high-fidelity archaeal DNA polymerase buffer.

[0023] In a twelfth embodiment, a method for carrying out a loop-mediated amplification reaction is provided. The method comprises two steps. The first step is to form a mixture comprising a nucleic acid template; four blocked cleavable primers forming a double helix with the nucleic acid template, which is a substrate for an RNase H2 protein; an RNase H2 protein selected from Q48R SEL29 (SEQ ID NO: 18) or another RNase H2 protein; a DNA polymerase protein; dNTP; and a buffer. The second step is to carry out an isothermal amplification cycle with the mixture.

[0024] In a thirteenth embodiment, a method is provided for performing an rhPCR assay in which primer dimer formation is reduced. The method includes the step of performing primer extension with Q48R SEL29 RNase H2 (SEQ ID NO: 18). The reduction in primer dimer formation corresponds to a reduction in the amount of primer dimers formed during an rhPCR assay with Q48R SEL29 RNase H2 (SEQ ID NO: 18) compared to an rhPCR assay performed with wild-type PaRNase H2 (SEQ ID NO: 1).

[0025] In a fourteenth embodiment, a method is provided for performing an rhPCR assay with improved mapping and on-target rates for a desired product. The method includes the step of performing primer extension with Q48R SEL29 RNase H2 (SEQ ID NO: 18). The improvement in mapping and on-target rates corresponds to an increase in mapping and on-target amplification of the desired product formed during the rhPCR assay with Q48R SEL29 RNase H2 (SEQ ID NO: 18) compared to an rhPCR assay performed with wild-type PaRNase H2 (SEQ ID NO: 1). [Brief explanation of the drawing]

[0026] [Figure 1A] This figure illustrates an exemplary plot showing that the dimerization rate when using the mutant Q48R SEL29 RNase H2 enzyme was lower at all enzyme concentrations compared to the wild-type PaRNase H2 enzyme. [Figure 1B] This figure illustrates illustrative data showing that the mapping rate of the mutant Q48R SEL29 RNase H2 enzyme is higher than that of the wild-type PaRNase H2 enzyme at all RNase H2 enzyme concentrations. [Figure 1C] This figure illustrates illustrative data showing that the on-target rate with the mutant Q48R SEL29 RNase H2 enzyme is higher than that of the wild-type PaRNase H2 enzyme at all enzyme concentrations. [Figure 2] This figure illustrates an example of the mean normalized dimer count per identified primer pair, in which case the mutant Q48R SEL29 RNase H2 enzyme reduces the highest number of identified primer dimers to half compared to the wild-type PaRNase H2 enzyme. [Figure 3A] This figure illustrates exemplary data regarding amplification product uniformity ≥0.2x and ≤0.05x (dropout rate). Library yields are equivalent for all concentrations between the mutant Q48R SEL29 RNase H2 enzyme and the wild-type PaRNase H2 enzyme. [Figure 3B] This figure illustrates that the overall amplification product homogeneity of the mutant Q48R SEL29 RNase H2 enzyme is equivalent to that of the wild-type PaRNase H2 enzyme at a range of test concentrations, with a ratio of ≥0.2. [Figure 3C] This figure illustrates that the dropout rate of the amplified product is similar for all tested titration concentrations between the mutant Q48R SEL29 RNase H2 enzyme and the wild-type PaRNase H2 enzyme. [Figure 4] This figure illustrates exemplary data showing that the homogeneity distribution (the percentage of amplified products with coverage within the ranges of 0-0.1x, 0.1-0.2x, 0.2x-0.5x, 0.5x-1.5x, 1.5x-2.5x, and 2.5x-5x compared to the mean coverage of amplified products) is equivalent between the mutant Q48R SEL29 RNase H2 enzyme and the wild-type PaRNase H2 enzyme. [Modes for carrying out the invention]

[0027] This invention provides novel hybrid RNase H2 enzyme variants that enhance enzymatic activity during rhPCR using a specific DNA polymerase buffer while retaining or enhancing the ability to recognize mismatches within a double-strand template. The RNase H2 enzyme hybrids combine amino acid sequence fragments derived from the organisms Pyrococcus abyssi (Pa), Thermocox kodacarensis (T. kod), and Pyrococcus fliosus. The resulting hybrid RNase H2 enzymes, as well as selected mutants based on these enzymes, exhibit markedly enhanced mismatch recognition. In particular, Q48R SEL29 RNase H2 (SEQ ID NO: 18) is shown to produce a mixed product with a reduced population of primer dimer species compared to a mixed product produced with wild-type PaRNase H2 (SEQ ID NO: 1) in methods for primer extension, rhPCR, target DNA sequence amplification, massively parallel sequencing, detection of SNP-containing nucleic acid templates from a nucleic acid template amplification product library, and loop-mediated amplification reactions.

[0028] definition To aid in understanding the present invention, several terms are defined below.

[0029] In the context describing the present invention (in particular in the context of the following claims), “a,” “an,” “it,” and similar references shall be understood to refer to both singular and plural forms unless otherwise indicated herein, or unless the context suggests otherwise. The terms “comprising,” “having,” “including,” and “containing” shall be interpreted as open-ended terms (i.e., “including but not limited to”) unless otherwise noted herein. The enumeration of value ranges herein is intended solely as a means of individually referring to each individual value that falls within the range unless otherwise indicated herein, and each individual value is incorporated herein as it would be if individually enumerated herein. All methods described herein may be performed in any appropriate order unless otherwise indicated herein, or unless the context suggests otherwise. The use of any or all examples or illustrative expressions presented herein (e.g., "etc.") is intended solely to better illustrate the invention and not to limit the scope of the invention as otherwise claimed. No expression herein should be understood to indicate that any unclaimed element is essential to the practice of the invention.

[0030] As used herein, the terms “nucleic acid” and “oligonucleotide” refer to any other type of polynucleotide that is a polydeoxyribonucleotide (containing 2-deoxy-D-ribose), a polyribonucleotide (containing D-ribose), or an N-glycoside of a purine or pyrimidine base. No distinction of length is intended between the terms “nucleic acid,” “oligonucleotide,” and “polynucleotide,” and these terms are used interchangeably. These terms refer only to the primary structure of the molecule. Therefore, these terms include double-stranded DNA and single-stranded DNA, as well as double-stranded RNA and single-stranded RNA. For use in the present invention, oligonucleotides may also include nucleotide analogs modified with bases, sugars, or phosphate backbones, as well as non-purine nucleotide analogs or non-pyrimidine nucleotide analogs.

[0031] Each oligonucleotide can be prepared by any suitable method, including direct chemical synthesis by methods such as the phosphodiester method according to Narang et al., 1979, Meth. Enzymol., 68:90-99; the phosphodiester method according to Brown et al., 1979, Meth. Enzymol., 68:109-151; the diethylphosphoramidite method according to Beaucage et al., 1981, Tetrahedron Lett., 22:1859-1862; and the solid-support method according to U.S. Patent No. 4,458,066, each incorporated herein by reference. A review of methods for synthesizing oligonucleotide conjugates and modified nucleotides is presented in Goodchild, 1990, Bioconjugate Chemistry, 1(3):165-187, incorporated herein by reference.

[0032] As used herein, the term “primer” refers to an oligonucleotide capable of acting as a starting point for DNA synthesis under appropriate conditions. Such conditions include conditions in which the synthesis of a primer elongation product complementary to the nucleic acid chain is induced, in the presence of appropriate buffer and temperature, four different nucleoside triphosphates, and an elongation agent (e.g., DNA polymerase or reverse transcriptase). Primer elongation may also be performed in the absence of one or more nucleotide triphosphates, in which case an elongation product of limited length is produced. As used herein, the term “primer” is intended to encompass oligonucleotides used in ligation-mediated reactions in which one oligonucleotide is “elongated” by ligation with a second oligonucleotide that hybridizes at an adjacent position. Thus, as used herein, the term “primer elongation” refers to both the polymerization of individual nucleoside triphosphates and the ligation of two oligonucleotides to form an elongation product, using the primer as a starting point for DNA synthesis.

[0033] The primer is preferably single-stranded DNA. The appropriate length of the primer depends on the intended use of the primer, but is typically in the range of 6 to 50 nucleotides, preferably 15 to 35 nucleotides. Shorter primer molecules generally require low temperatures to form a sufficiently stable hybrid complex with the template. The primer does not need to reflect the exact sequence of the template nucleic acid, but it must be sufficiently complementary to the template for hybridization. In the art, primer designs suitable for amplification are well known and described in the literature cited herein.

[0034] Primers may incorporate additional features that enable detection or immobilization of the primer but do not alter the primer's fundamental property of acting as a starting point for DNA synthesis. For example, a primer may contain an additional nucleic acid sequence at its 5' end that does not hybridize with the target nucleic acid but facilitates the cloning or detection of the amplified product. In this specification, a region of the primer that is sufficiently complementary to the template to the extent that it hybridizes is referred to as the hybridizing region.

[0035] As used herein, the terms “target,” “target sequence,” “target region,” and “target nucleic acid” are synonymous and refer to a region or sequence of nucleic acid that is amplified, sequenced, or detected.

[0036] As used herein, the term “hybridization” refers to the formation of a double-stranded structure from two single-stranded nucleic acids resulting from complementary base pairing. Hybridization may occur between perfectly complementary nucleic acid strands or between “substantially complementary” nucleic acid strands containing small mismatch regions. Hybridization of perfectly complementary nucleic acid strands is strongly preferred under conditions referred to as “strict hybridization conditions” or “sequence-specific hybridization conditions.” Stable double helix formation from substantially complementary sequences can be achieved under less strict hybridization conditions; however, the acceptable degree of mismatch is controlled by appropriate adjustment of the hybridization conditions. Those skilled in the field of nucleic acid technology may determine the stability of a double helix by empirically considering a number of variables, including, for example, the length and base pair composition of oligonucleotides, ionic strength, and the occurrence of mismatched base pairs, in accordance with guidelines provided by the art (see, for example, Sambrook et al., 1989, "Molecular Cloning - A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol., 26(3 / 4):227~259; and Owczarzy et al., 2008, Biochemistry, 47:5336~5353, which are incorporated herein by reference).

[0037] The term “amplification reaction” refers to any chemical reaction, including enzymatic reactions, that results in an increase in the number of copies of a template nucleic acid sequence or in the transcription of a template nucleic acid. Amplification reactions include polymerase chain reaction (PCR) and ligase chain reaction (LCR) (see Barany et al., U.S. Patent No. 5,494,810), including reverse transcription and real-time PCR (see U.S. Patents No. 4,683,195 and 4,683,202; see "PCR Protocols: A Guide to Methods and Applications" (Innis et al., 1990)). Exemplary “amplification reaction conditions” or “amplification conditions” typically include a two-step cycle or a three-step cycle. A two-step cycle has a hybridization / extension (or ligation) step following a high-temperature denaturation step. A three-step cycle has a separate extension or ligation step following a hybridization step following a denaturation step.

[0038] As used herein, "polymerase" refers to an enzyme that catalyzes the polymerization of nucleotides. Generally, the enzyme initiates synthesis at the 3' end of a primer annealed to a nucleic acid template sequence. "DNA polymerase" catalyzes the polymerization of deoxyribonucleotides.Known DNA polymerases include, for example, Pyrococcus fryosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108:1), E. coli DNA polymerase I (Lecomte and Doubleday, 1983, Nucleic Acids Res., 11:7505), T7 DNA polymerase (Nordstrom et al., 1981, J. Biol. Chem., 256:3112), Thermus thermophilus (Tth) DNA polymerase (Myers and Gelfand, 1991, Biochemistry, 30:7661), and Bacillus stearothermophilus DNA polymerase (Stenesh and McGowan, 1977, Biochim Biophys). Acta, 475:32), Thermococcus litoralis (Tli) DNA polymerase (also called Vent DNA polymerase; Cariello et al., 1991, Nucleic Acids Res., 19:4193), Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998, Braz J. Med. Res, 31:1239), Thermus aquaticus (Taq) DNA polymerase (Chien et al., 1976, J. Bacteoriol, 127:1550), Thermococcus kodakaraensis (KOD) This includes DNA polymerase (Takagi et al., 1997, Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase (Patent Application No. WO0132887), and Pyrococcus GB-D (PGB-D) DNA polymerase (Juncosa-Ginesta et al., 1994, Biotechniques, 16:820). The polymerase activity of any of the above enzymes can be determined by means known in the art.

[0039] The primers used herein are "specific" to the target sequence if, when used in amplification reactions under sufficiently strict conditions, they primarily hybridize to the target nucleic acid. Typically, a primer is specific to a target sequence if the stability of the primer-target duplex is greater than the stability of the duplex formed between the primer and any other sequence found in the sample. Those skilled in the art will recognize that various factors, including salt conditions, primer base composition, and mismatch locations, affect primer specificity, and that in many cases, confirmation of primer specificity by conventional experiments is necessary. Hybridization conditions can be selected that allow the primer to form a stable duplex only with the target sequence. Therefore, the use of target-specific primers under appropriately strict amplification conditions enables selective amplification of the target sequence containing the target primer-binding site.

[0040] As used herein, the term “non-specific amplification” refers to the amplification of nucleic acid sequences other than the target sequence, resulting from a primer that hybridizes with a sequence other than the target sequence and is subsequently used as a substrate for primer extension. Hybridization of primers with non-target sequences is referred to as “non-specific hybridization” and tends to occur particularly at low temperatures, low strictness, in the pre-amplification state, or when a mutant allele with a sequence very closely related to the true target is present in the sample, as in the case of single nucleotide polymorphisms (SNPs).

[0041] The term "3' mismatch identification" refers to the property of DNA polymerase to distinguish a fully complementary sequence from a mismatch-containing (nearly complementary) sequence in which the extended nucleic acid (e.g., a primer or other oligonucleotide) has a mismatch at its 3' end compared to the template into which the nucleic acid hybridizes. In some embodiments, the extended nucleic acid contains a mismatch at its 3' end compared to a fully complementary sequence.

[0042] The term "3'-side mismatch identification assay" refers to an assay that identifies the presence of improved specificity in amplification of a target DNA sequence when the target DNA sequence is examined by two primers that have substantially identical sequences except for the occurrence of one or more nucleotide residues with different base compositions at or near their respective 3' ends. For example, a first primer having a 3' end sequence that is perfectly complementary to the target DNA sequence is considered a 3'-side match primer, while a second primer having a 3' end sequence with at least one nucleotide base that is incomplementary to the target DNA sequence is considered a 3'-side mismatch primer. Examples of 3'-side mismatch identification assays are presented in many of the examples, particularly in Tables 10 and 11 of Example 4.

[0043] As used herein, the term “primer dimer” refers to a template-independent, nonspecific amplification product that is thought to arise from primer extension, in which case another primer is used as a template. Primer dimers are often concatamers, i.e., dimers, of two primers, but concatamers of more than two primers can also occur. In this specification, the term “primer dimer” is used to generally encompass template-independent, nonspecific amplification products.

[0044] As used herein, the term “reaction mixture” refers to a solution containing the reagents necessary to carry out a given reaction. “Amplification reaction mixture,” which refers to a solution containing the reagents necessary to carry out an amplification reaction, typically contains oligonucleotide primers and DNA polymerase or DNA ligase in a suitable buffer. “PCR reaction mixture” typically contains oligonucleotide primers, DNA polymerase (most typically, thermally stable DNA polymerase), dNTPs, and divalent metal cations in a suitable buffer. A reaction mixture is referred to as complete if it contains all the reagents necessary to enable the reaction, and as incomplete if it contains only a subset of the required reagents. Those skilled in the art will understand that the reaction components are typically stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for adjustment of component concentrations depending on the application, and that the reaction components are combined before the reaction to create a complete reaction mixture. Furthermore, it will be understood by those skilled in the art that the reactive components are individually packaged, and a useful commercial kit may contain any subset of the reactive components, including the protective primer of the present invention.

[0045] For the purposes of the present invention, the terms “inactivated” or “deactivated” as used herein refer to a primer or other oligonucleotide that is unable to participate in a primer extension or ligation reaction because DNA polymerase or DNA ligase cannot interact with the oligonucleotide for their intended purpose. In some embodiments, where the oligonucleotide is the primer, the inactivated state occurs because the primer is protected at or near its 3' end to prevent primer extension. If a specific group is bound at or near the 3' end of the primer, DNA polymerase cannot bind to the primer and extension cannot occur. However, an inactivated primer can hybridize with a substantially complementary nucleotide sequence.

[0046] For the purposes of the present invention, the term “activation” as used herein refers to a primer or other oligonucleotide capable of participating in a reaction with DNA polymerase or DNA ligase. The primer or other oligonucleotide is activated after hybridizing with a substantially complementary nucleic acid sequence and is cleaved to yield a functional 3' or 5' end so that it can interact with DNA polymerase or DNA ligase. For example, if the oligonucleotide is a primer and the primer hybridizes with a template, the 3' blocking group is removed from the primer, for example, by a cleaving enzyme, so that DNA polymerase can bind to the 3' end of the primer and facilitate primer elongation.

[0047] As used herein, the terms “cleavage domain” or “cleavage domain” are synonymous and refer to a region located between the 5' and 3' ends of a primer or other oligonucleotide, recognized by a cleavage compound, such as a cleavage enzyme, that cleaves the primer or other oligonucleotide. For the purposes of the present invention, the cleavage domain is designed to be cleaved only when the primer or other oligonucleotide is hybridized with a complementary nucleic acid sequence, and not when it is single-stranded. The cleavage domain or adjacent sequences may include: a) a portion that blocks or inhibits the extension or ligation of the primer or other oligonucleotide by a polymerase or ligase; b) a portion that enhances the identification of mutant alleles; or c) a portion that suppresses unwanted cleavage reactions. One or more such portions may be contained within the cleavage domain or within adjacent sequences.

[0048] As used herein, the term “RNase H cleavage domain” refers to a type of cleavage domain containing one or more ribonucleic acid residues or alternative analogues that provide a substrate for RNase H. The RNase H cleavage domain can be positioned at any location within the primer or oligonucleotide, preferably at or near the 3' end of the molecule, or at the 5' end.

[0049] The "RNase H2 cleavage domain" may contain a single RNA residue, a sequence of adjacently linked RNA residues, or RNA residues separated by a DNA residue or another chemical group. In one embodiment, the RNase H2 cleavage domain is a 2'-fluoronucleoside residue. In a more preferred embodiment, the RNase H2 cleavable domain contains two adjacent 2'-fluoro residues.

[0050] As used herein, the term “protecting primer” refers, at a minimum, to a primer having a target sequence and a cleavable domain suitable for sufficient hybridization, a cleavable domain, and a blocking group that prevents extension from the 3' end of the primer until cleavage occurs. In a preferred embodiment, the cleavable domain is an RNase H cleavage domain, and the blocking group is a propanediol (C3) spacer.

[0051] As used herein, the terms “cleavage compound” or “cleavage agent” refer to any compound that recognizes a cleavage domain within a primer or other oligonucleotide and selectively cleaves the oligonucleotide based on the presence of the cleavage domain. The cleavage compounds used in the present invention selectively cleave the primer or other oligonucleotide containing the cleavage domain only when hybridized with a substantially complementary nucleic acid sequence, and do not cleave the primer or other oligonucleotide when single-stranded. The cleavage compound cleaves the primer or other oligonucleotide that is within or adjacent to the cleavage domain. As used herein, the term “adjacent” means that the cleavage compound cleaves the primer or other oligonucleotide at the 5' or 3' end of the cleavage domain. The preferred cleavage reactions in the present invention result in a 5'-phosphate group and a 3'-OH group.

[0052] In a preferred embodiment, the cleavage compound is a “cleavage enzyme.” A cleavage enzyme is a protein or ribozyme that can recognize a cleavage domain when a primer or other nucleotide is hybridized with a substantially complementary nucleic acid sequence, but does not cleave the complementary nucleic acid sequence (i.e., results in a single-strand break within a double helix). A cleavage enzyme also does not cleave a primer or other oligonucleotide containing a cleavage domain if it is single-stranded. Examples of cleavage enzymes are RNase H enzymes and other nicking enzymes.

[0053] As used herein, the term “nicking” refers to the cleavage of only one strand of a double-stranded portion of a fully double-stranded nucleic acid or a partially double-stranded nucleic acid. The site where a nucleic acid is nicked is referred to as the “nicking site” (NS). A “nicking agent” (NA) is an agent that partially or completely nicks a double-stranded nucleic acid. The NA may be an enzyme, or any other compound or composition. In certain embodiments, the nicking agent may recognize a specific nucleotide sequence of a fully double-stranded nucleic acid or a partially double-stranded nucleic acid and cleave only one strand of the fully double-stranded nucleic acid or a partially double-stranded nucleic acid at a specific site (i.e., the NS) compared to the site of the recognition sequence. Such nicking agents (referred to as “sequence-specific nicking agents”) include, but are not limited to, nicking endonucleases (e.g., N.BstNB).

[0054] Therefore, as used herein, "nicking endonuclease" (NE) refers to an endonuclease that recognizes the nucleotide sequence of a fully double-stranded or partially double-stranded nucleic acid molecule and cleaves only one strand of the nucleic acid molecule at a specific position compared to the recognition sequence. In such cases, the entire sequence from the recognition site to the cleavage site constitutes the "cleavage domain".

[0055] As used herein, the term “blocking group” refers to a chemical moiety bound to a primer or other oligonucleotide in such a way that amplification does not occur. For example, primer extension and / or DNA ligation does not occur. Once the blocking group is removed from the primer or other oligonucleotide, the oligonucleotide can participate in the assay for which it was designed (PCR, ligation, sequencing, etc.). Therefore, a “blocking group” can be any chemical moiety that inhibits recognition by polymerase or DNA ligase. Blocking groups can generally be incorporated into cleavage domains located on the 5' or 3' side of the cleavage domain. A blocking group can consist of one or more chemical moieties. In the present invention, “blocking groups” are typically removed after hybridization of the oligonucleotide with its target sequence.

[0056] The term "fluorescence-generating probe" refers to a) oligonucleotides conjugated with a fluorophore and a quencher, and optionally, a sub-groove binder or b) a DNA-binding reagent such as SYBR® Green dye.

[0057] The terms "fluorescent label" or "fluorophore" refer to compounds whose maximum fluorescence emission is between approximately 350 and 900 nm. Examples include: 5-FAM (also known as 5-carboxyfluorescein; also known as Spiro(isobenzofuran-1(3H),9'-(9H)xanthene)-5-carboxylic acid, 3',6'-dihydroxy-3-oxo-6-carboxyfluorescein); 5-hexachlorofluorescein; ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceinyl)-6-carboxylic acid]); 6-H Xachlorofluorescein; ([4,7,2',4',5',7'-Hexachloro-(3',6'-Dipivaloylfluoresceinyl)-5-carboxylic acid]); 5-Tetrachlorofluorescein; ([4,7,2',7'-Tetra-chloro-(3',6'-Dipivaloylfluoresceinyl)-5-carboxylic acid]); 6-Tetrachlorofluorescein; ([4,7,2',7'-Tetrachloro-(3',6'-Dipivaloylfluoresceinyl)-5-carboxylic acid]); Xanthylium is Xanthylium, which is Xenyl (6-6-carboxytetramethylrhodamine), 9-(2,4-dicarboxyphenyl)-3,6-bis(dimethylamino), and EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid). A wide variety of fluorophores can be used, including but not limited to 1,5-IAEDANS(5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid); Cy5(indodicarbocyanine-5); Cy3(indodicarbocyanine-3); and BODIPYFL(2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid); Quasar®-670 dye (Biosearch Technologies); Cal Fluor® Orange dye (Biosearch Technologies); Rox dye; Max dye (Integrated DNA Technologies), as well as suitable derivatives thereof.

[0058] As used herein, the term “quencher” refers to a molecular or compound portion capable of reducing emission from a fluorescent donor when bound to or in close proximity to a donor. Quenching can occur through any of several mechanisms, including excitation coupling such as fluorescence resonance energy transfer, photo-induced electron transfer, paramagnetic intersystem cross-enhancement, Dexter exchange coupling, and dark complex formation. Fluorescence is “quenched” if the fluorescence emitted by the fluorophore is reduced by at least 10%, for example, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9%, compared to the fluorescence in the absence of the quencher. In this technical field, numerous commercially available quenchers are known, including, but not limited to, DABCYL, BlackHole® Quenchers (BHQ-1, BHQ-2, and BHQ-3), Iowa Black® FQ, and Iowa Black® RQ. These are so-called dark quenchers. They do not have natural fluorescence and effectively eliminate the background problems associated with other quenchers, such as TAMRA, which is intrinsically fluorescent.

[0059] As used herein, the term “ligation” refers to the covalent linkage of two polynucleotide ends. In various embodiments, ligation involves a covalent linkage of the 3' end of a first polynucleotide (acceptor) to the 5' end of a second polynucleotide (donor). Ligation results in the formation of a phosphodiester bond between the polynucleotide ends. In various embodiments, ligation may be mediated by any enzyme, chemical reaction, or process that results in the covalent linkage of the polynucleotide ends. In certain embodiments, ligation is mediated by a ligase enzyme.

[0060] As used herein, "ligase" refers to an enzyme capable of covalently linking the 3' hydroxyl group of one polynucleotide to the 5' phosphate group of a second polynucleotide. Examples of ligases include E. Corley DNA ligase and T4 DNA ligase.

[0061] Ligation reactions can also be used in DNA amplification methods such as ligase chain reactions (LCR), also known as ligase amplification reactions (LAR) (see also Barany, Proc. Natl. Acad. Sci., 88:189 (1991); and Wu and Wallace, Genomics, 4:560 (1989), incorporated herein by reference). In an LCR, four oligonucleotides are mixed, each being a complementary set of two adjacent oligonucleotides that hybridize specifically to one strand of the target DNA and an adjacent oligonucleotide that hybridizes to the opposite strand. DNA ligase is then added to the mixture. In the presence of the target sequence, the DNA ligase covalently links each set of hybridized molecules. Importantly, in an LCR, two oligonucleotides are ligated together only if they base-pair with the sequence without gaps. The repetition of the denaturation, hybridization, and ligation cycle amplifies short segments of DNA. Mismatches at the junctions between adjacent oligonucleotides inhibit ligation. In other oligonucleotide ligation assays, this property allows LCRs to be used to distinguish mutant alleles such as SNPs. LCRs have also been used in combination with PCR to achieve enhanced detection of single nucleotide changes (see Segev, PCT Publication WO9001069 (1990)).

[0062] The term "codon optimization" refers to the modification of specific nucleic acids encoding polypeptides to incorporate codons that are favorable for efficient expression within a given host cell, such as a given microbial cell (e.g., E. coli, S. cerevisae) or mammalian cell (e.g., human cells such as HeLa cells and COS cells). In the art, such favorable codons are well known based on codon bias tables developed for various organisms. The polynucleotides encoding polypeptides of the present invention include polynucleotides having a codon-optimized open reading frame for any known organism for which a codon bias table has been developed, or for which such a table can be readily identified by empirical determination.

[0063] The term "BaseX-based PCR amplification" refers to a highly efficient nucleic acid amplification method that enables more than a twofold increase in the amplification product for each amplification cycle, thereby enabling increased sensitivity and speed compared to conventional PCR. This method is disclosed in U.S. Patent Publication US10273534(B2), published on April 30, 2019, by R. Higuchi (applicant: Cepheid), titled "Exponential base-greater-than-2 nucleic acid amplification," which is incorporated herein by reference in its entirety.

[0064] The terms "fusion protein" or "fusion polypeptide" refer to the incorporation of non-native amino acid information into a protein, where additional amino acid information is covalently linked. Such additional amino acid information may include tags that enable the purification or identification of the fusion protein. Such additional amino acid information may include peptides that enable the fusion protein to be transported into and / or to specific locations within the cell. Examples of tags for these purposes include: AviTag, a peptide that can be isolated by streptavidin because it enables biotinylation by the enzyme BirA; Calmodulin tag, a peptide that is bound by the protein calmodulin; Polyglutamic acid tag, a peptide that efficiently binds to anion exchange resins such as Mono-Q; E tag, a peptide recognized by an antibody; FLAG tag, a peptide recognized by an antibody; HA tag, a peptide derived from hemagglutinin recognized by an antibody; typically, nickel chelating agents or cobalt. His tag: 5-10 histidines conjugated with a chelating agent; Myc tag: A peptide derived from c-myc recognized by an antibody; NE tag: A novel 18-amino acid synthetic peptide recognized by monoclonal IgG1 antibody, useful in a wide range of applications including Western blotting, ELISA, flow cytometry, immunohistochemistry, immunoprecipitation, and recombinant protein affinity purification; S tag: A peptide derived from ribonuclease A; SBP tag: A peptide that binds to streptavidin; Softag tag: Intended for expression in mammals. 1; Softag, intended for expression in prokaryotes; 3; Strep tag, a peptide that binds to modified streptavidin called streptavidin or streptactin (Strep tag II); TC tag, an arsenic tetracysteine ​​tag recognized by FlAsH arsenic compound and ReAsH arsenic compound; V5 tag, an antibody-recognized peptide; VSV tag, an antibody-recognized peptide; Xpress tag; Isopeptag, a peptide that covalently binds to pyrin C protein;The tags include: SpyTag, a peptide that covalently binds to the SpyCatcher protein; SnoopTag, a peptide that covalently binds to the SnoopCatcher protein; BCCP (biotin carboxyl carrier protein), a protein domain biotinylated by BirA to enable recognition by streptavidin; glutathione-S-transferase tag, a protein that binds to immobilized glutathione; green fluorescent protein tag, a protein that is spontaneously fluorescent and can be bound by antibodies; HaloTag, a mutant bacterial haloalkane dehalogenase that covalently binds to reactive haloalkane substrates, enabling binding to a wide variety of substrates; maltose-binding protein tag, a protein that binds to amylose agarose; Nus tag; thioredoxin tag; and an Fc tag derived from an immunoglobulin Fc domain that enables dimerization and solubilization and can be used for purification on protein A Sepharose. Nuclear localization signals (NLS), such as NLS obtained from SV40, enable proteins to be transported to the nucleus immediately after entering a cell. Given that the natural Cas9 protein, being of bacterial origin, does not naturally contain NLS motifs, the addition of one or more NLS motifs to the recombinant Cas9 protein is expected to improve genome editing activity when used in eukaryotic cells where the target genome's DNA substrate is constitutively located in the nucleus. Those skilled in the art will understand these diverse fusion tag technologies, the specific amino acid sequences involved, and how to construct and use fusion proteins containing them. In one embodiment, a highly preferred fusion protein or fusion polypeptide contains the His tag motif, but those skilled in the art will understand that other tags may also be included, as mentioned above. In addition to fusion proteins or fusion polypeptides, the present invention includes original mutant forms of the corresponding proteins or polypeptides lacking further amino acid sequence information.

[0065] Novel hybrid RNase H2 enzyme variants created by recombinant reshuffling of amino acid sequences derived from known RNase H2 enzymes. Two hybrid RNase H2 enzymes with novel and useful properties can be created through partial recombination ("shuffling") of amino acid sequences derived from three RNase H2 enzymes, including Pirococcus abyssi, Thermococcus kodacarensis, and Pirococcus phryosus, resulting in hybrid RNase H2 enzymes. In particular, two mutant RNase H2 enzymes, SEL28 RNase H2 (SEQ ID NO: 89) and SEL29 RNase H2 (SEQ ID NO: 90), which combine amino acid sequence fragments from the organisms Pirococcus abyssi, Thermococcus kodacarensis, and Pirococcus phryosus, were found to significantly enhance mismatch recognition. The mutants were selected from a library created by random shuffling of RNase H2 sequences. Other variations also exist, but all mutant enzymes contain fragments of amino acid residues 26-40 and 100-120 of T.kod RNase H2. Based on homology with known crystal structures (Muroya et al., 2001; Rychlik et al., 2010), these residues are likely to be in contact with the bound DNA double helix. Independent of any particular theory, it can be hypothesized that residues 26-40 and 100-120 of T.kod RNase H2 alter the enzyme binding pocket to the substrate double helix, resulting in changes in binding affinity and nucleic acid cleavage catalysis. The amino acid sequences of wild-type P.ab.RNase H2 protein (SEQ ID NO: 88), hybrid SEL28 RNase H2 protein (SEQ ID NO: 89), and hybrid SEL29 RNase H2 protein (SEQ ID NO: 90) are shown in Table 1. The corresponding (His)6-tagged amino acid sequences for the wild-type P.ab.RNase H2 protein (SEQ ID NO: 1), the hybrid SEL28 RNase H2 protein (SEQ ID NO: 2), and the hybrid SEL29 RNase H2 protein (SEQ ID NO: 3) were also prepared and used as a base for creating further mutant RNase H2 proteins (see Table 3).

[0066] [Table 1] TIFF0007900392000003.tif133166

[0067] The resulting hybrid SEL28 RNase H2 and SEL29 RNase H2 enzymes, encoded by SEQ ID NOs. 89 and 90, improve mismatch recognition when the mismatch is located on an RNA nucleotide, but exhibit varying degrees of specificity (data not shown). Similarly, the SEL28 mutant RNase H2 and SEL29 mutant RNase H2 enzymes, encoded by SEQ ID NOs. 89 and 90, improve mismatch recognition at the 5' end of the RNA nucleotide (data not shown).

[0068] rhPCR could also be performed using high-fidelity DNA polymerases (such as DNA polymerases derived from Pyrococcus phlosus and Thermocox kodacarensis (KOD)) instead of DNA polymerases derived from Thermus aquatics (Taq). However, WT PaRNase H2, SEL28 RNase H2, and SEL29 RNase H2 have limited activity in rhPCR using high-fidelity polymerases and associated reaction buffers. The optimal reaction buffer for Taq DNA polymerase is substantially different from the optimal buffer for KOD DNA polymerase. Mutations in RNase H2 that are more tolerant of components in the reaction buffer for KOD DNA polymerase may be found. The inventors demonstrate that Q48R, A107V, and P13S / A107V enhance enzyme activity using KOD DNA polymerase reaction buffer when added to the hybrid mutants SEL28 RNase H2 or SEL29 RNase H2.

[0069] This invention relates to mutant RNase H2 enzymes that enhance enzyme activity during rhPCR using KOD DNA polymerase and its optimal reaction buffer. Mutations at amino acids 48 and 107 of SEL29 RNase H2 have been shown to improve this activity. Screening of seven point mutants with a background of SEL28 RNase H2 or SEL29 RNase H2 showed that Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 enhance enzyme activity using KOD DNA polymerase reaction buffer. They were also shown to retain or enhance the mismatch recognition ability of SEL29 RNase H2.

[0070] RNase H2-mediated PCR The hybrid RNase H2 mutant proteins disclosed herein can be used in a variety of PCR applications. RNase H2-dependent PCR is a method that increases PCR specificity and eliminates primer dimers by using RNase H2 derived from Pyrococcus abyssi or related organisms, along with DNA primers ("blocked cleavable primers") containing a single ribonucleotide residue and a 3' protective region. Blocked cleavable primers are activated when cleaved by the RNase H2 enzyme. The cleavage occurs at the 5' end of the RNA base after hybridization with the target DNA. Since primers can only be cleaved after hybridization with a perfectly matched target sequence, primer dimers are reduced. The demand for high target complementarity reduces the amplification of closely related sequences.

[0071] In this regard, hybrid RNase H2 mutant proteins are particularly suitable for enhancing performance in producing high-quality genome amplification product libraries for high-throughput multiplex sequencing applications, such as next-generation sequencing (NGS). In particular, Q48R SEL29 is an RNase H2 enzyme useful for RNase H2-mediated PCR applications and systems such as our rhAmpSeq™ system. RNase H2-mediated SNP detection and RNase H2-mediated rare allele detection To enhance the mismatch identification attribute at the 3' end, the hybrid RNase H2 mutant protein disclosed herein can be used to detect single nucleotide polymorphisms and rare alleles. The use of blocked cleavable primers that form a complete double helix with the desired SNP-containing nucleic acid template is recognized and cleaved by the hybrid RNase H2 mutant protein, thereby activating the primer-desired nucleic acid template double helix for primer extension by DNA polymerase under appropriate conditions.

[0072] RNase H2 in loop-mediated isothermal amplification (LAMP) In this specification, the use of RNase H2 in loop-mediated isothermal amplification (LAMP) is also assumed. The LAMP amplification method is carried out under isothermal conditions without changes in reaction temperature during cycling. LAMP requires a minimum of four different primers designed to recognize six different regions of the desired unit replication sequence (Notomi et al., Nucleic Acids Research, 28(12)(2000)). The amplification reaction typically relies on the strand displacement activity of DNA polymerase derived from Bacillus stearotermophilus (Bst). The product has a structure consisting of long-chain reverse repeats of the target sequence.

[0073] The LAMP reaction is prone to primer dimerization due to the use of numerous primers and mesothermic DNA polymerases in the method. LAMP also lacks 5'→3' exonuclease activity in BST polymerase amplification, due to the fact that this activity disrupts amplification by competing with essential strand displacement activity. The use of blocked cleavable primers and RNase H2 can be utilized to reduce or eliminate the detection of primer dimer signals in the LAMP reaction. In this regard, hybrid RNase H2 mutant proteins are particularly suitable for enhancing the performance of desired products formed in the LAMP reaction without the associated generation of primer dimers.

[0074] Applicable In a first embodiment, a hybrid RNase H2 protein is provided. The hybrid RNase H2 protein contains amino acid sequence fragments derived from the organisms Pyrococcus abyssi (Pa), Thermocox kodacarensis (T. kod), and Pyrococcus phryosus. In the first related matter, the hybrid RNase H2 protein contains amino acid residues 26-40 and 100-120 of T. kod RNase H2. In the second related matter, the hybrid RNase H2 protein is selected from SEQ ID NOs: 2 and 3. In the third related matter, the hybrid RNase H2 protein is selected from SEQ ID NOs: 14-20.

[0075] In a second embodiment, recombinant nucleic acids encoding any of the hybrid RNase H2 proteins disclosed herein are provided. In the first related matter, exemplary recombinant nucleic acids encoding any of the hybrid RNase H2 proteins include SEQ ID NOs. 79-87 in Table 14.

[0076] In a third embodiment, a method for performing primer extension is provided. The method comprises the step of contacting a hybrid RNase H2 protein, as disclosed herein, with a primer, a polynucleotide template, a nucleoside triphosphate, and a DNA polymerase, under conditions suitable for a primer extension method, thereby producing an extension primer. In the first related matter, the DNA polymerase includes high fidelity archaeal DNA polymerase. In the second related matter, the primer includes a blocked cleavable primer. In the third related matter, the primer extension method includes a method for performing a polymerase chain reaction (PCR). In the fourth related matter, the method for performing PCR improves mismatch recognition in the primer:polynucleotide hybrid formed between the primer and the polynucleotide template. In the fifth related matter, the improvement in mismatch recognition includes improvement in mismatch recognition at the 3' end.

[0077] In a fourth embodiment, a reaction mixture is provided. The reaction mixture comprises a hybrid RNase H2 protein, at least one primer, a polynucleotide template, a nucleoside triphosphate, and a DNA polymerase, as described herein. In the first relating matter, the reaction mixture comprises a DNA polymerase which is a high-fidelity archaeal DNA polymerase. In the second relating matter, the reaction mixture comprises at least one primer which is a blocked cleavable primer.

[0078] In a fifth aspect, a method for performing rhPCR is provided. The method comprises the step of performing primer extension with a hybrid RNase H2, a DNA polymerase, and a primer as described herein. In the first related matter, the method for performing rhPCR comprises the step of performing primer extension with a high-fidelity archaeal DNA polymerase. In the second related matter, the hybrid RNase H2 enzyme is reversibly inactivated by chemical modification, an aptamer, or a blocking antibody. In the third related matter, a blocking group is bound to the 3' terminal nucleotide of the primer. In the fourth related matter, a blocking group is bound to the 5' side of the 3' terminal residue, inhibiting the primer from being used as a template for DNA synthesis. In the fifth related matter, the blocking group comprises one or more debasic residues. In the sixth related matter, one or more debasic residues are C3 spacers. In relation to the seventh aspect, the blocking group comprises one member selected from the group consisting of RDDDDx, RDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM, and DxxD [in the sequence, R is an RNA residue, D is a DNA residue, M is a mismatch residue, and x is a C3 spacer or another patented group resistant to degradation]. In this respect, the blocking group comprises a label enabling detection of the extension amplification reaction. In this respect, the label enabling detection of the amplification reaction is bound to the oligonucleotide primer 3' from the cleavage site. In this respect, the label is a mass tag for detection of the amplification reaction by fluorophores or mass spectrometry. In a further embodiment, the cleavage domain of the blocked cleavable primer comprises one or more of the following: DNA residues, debased residues, modified nucleosides, or internucleotide modified phosphate linkages. In further related matters, cleavage domains include a single RNA residue, two adjacent RNA residues, or a sequence of three or more RNA residues, and lack an RNA residue or one or more 2' modified nucleosides.In relation to the cleavage domain containing one or more 2'-modified nucleosides, the one or more 2'-modified nucleosides are selected from the group consisting of 2'-O-alkylRNA nucleosides, 2'-fluoronucleosides, locked nucleic acids, 2'-ethylene nucleic acid residues, 2'-alkylnucleosides, 2'-aminonucleosides, and 2'-thionucleosides. Exemplary 2'-modified nucleosides include 2'-O-methylRNA nucleosides and 2'-fluoronucleosides.

[0079] A sixth embodiment provides a method for amplifying a target DNA sequence. The method comprises several steps. The first step is to prepare a reaction mixture comprising: (i) an oligonucleotide primer having a cleavage domain cleavable by an RNase H2 enzyme, located at the 5' end of a blocking group linked at or near the 3' end of the oligonucleotide primer, wherein the blocking group prevents primer elongation and / or inhibits the use of the oligonucleotide primer as a template for DNA synthesis; (ii) a sample nucleic acid, which may or may not be a target sequence; (iii) a DNA polymerase; and (iv) a hybrid RNase H2 protein disclosed herein. The second step is to hybridize the oligonucleotide primer with the target DNA sequence to form a double-stranded substrate. The third step is to remove the blocking group from the oligonucleotide primer by cleaving the hybridized oligonucleotide primer at a cleavage site within the cleavage domain or at a cleavage site adjacent to the cleavage domain using a hybrid RNase H2 enzyme. In the first related matter of the method, the DNA polymerase is an archaeal high-fidelity DNA polymerase. In the second related matter, the RNase H2 protein is reversibly inactivated by chemical modification or a blocking antibody. In a further related matter of the method, the blocking group is bound to the 3' terminal nucleotide of the oligonucleotide primer. In a further related matter of the method, the blocking group is bound to the 5' side of the 3' terminal residue, inhibiting the oligonucleotide primer from being used as a template for DNA synthesis. In a further related matter of the method, the blocking group comprises one or more debasing residues. In a further related matter of the method, one or more debasing residues are C3 spacers or other patented groups that are resistant to degradation.In further relating to the method, the blocking group comprises one member selected from the group consisting of RDDDDx, RDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM, and DxxD [in the sequence, R is an RNA residue, D is a DNA residue, M is a mismatch residue, and x is a C3 spacer or another patented group resistant to degradation]. In further relating to the method, the blocking group comprises a label enabling detection of the extension amplification reaction. In further relating to the method, the method further comprises a label enabling detection of the amplification reaction, in which case the label is bound to an oligonucleotide primer 3' from the cleavage site. In these relating matters, the label is a mass tag for detection of the amplification reaction by fluorophore or mass spectrometry. In further relating to the method, the cleavage domain comprises one or more of the following parts: a DNA residue, a debased residue, a modified nucleoside, or an internucleotide modified phosphate linkage. In further relating to the method, the cleavage domain includes a single RNA residue, two adjacent RNA residues, a sequence of three or more RNA residues, or one or more 2'-modified nucleosides. In relating to the cleavage domain including one or more 2'-modified nucleosides, these 2'-modified nucleosides are selected from the group consisting of 2'-O-alkylRNA nucleosides, 2'-fluoronucleosides, locked nucleic acids, 2'-ethylene nucleic acid residues, 2'-alkylnucleosides, 2'-aminonucleosides, and 2'-thionucleosides. Exemplary 2'-modified nucleosides include 2'-O-methylRNA nucleosides and 2'-fluoronucleosides.

[0080] In a seventh aspect, a kit for preparing extension primers is provided. The kit comprises at least one container providing a hybrid RNase H2 protein as disclosed herein. In the first aspect, the kit further comprises one or more further containers selected from the group consisting of (a) a container providing a primer that can hybridize with a given polynucleotide template under extension conditions; (b) a container providing a nucleoside triphosphate; (c) a container providing a buffer suitable for primer extension; and (d) a DNA polymerase. In the second aspect, the DNA polymerase comprises a high-fidelity archaeal DNA polymerase. In the third aspect, the kit comprises one or more further containers containing blocked cleavable primers.

[0081] In an eighth aspect, a kit for performing amplification of a target DNA sequence is provided. The kit comprises a reaction buffer comprising RNase H2 as described herein and a high-fidelity archaeal DNA polymerase. In the first aspect, the kit further comprises one or more oligonucleotide primers, at least one oligonucleotide primer having a cleavage domain cleavable by the RNase H2 enzyme, located at the 5' end of a blocking group linked at or near the 3' end of the oligonucleotide primer, the blocking group preventing primer elongation and / or inhibiting the use of the oligonucleotide primer as a template for DNA synthesis. In the second aspect, the kit comprises a blocking group that is one member selected from the group consisting of RDDDDx, RDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD [in the sequence, R is an RNA residue, D is a DNA residue, M is a mismatch residue, and x is a C3 spacer or another patented group resistant to degradation].

[0082] In a ninth aspect, a method for preparing an amplification product library of template nucleic acids is provided. The method comprises several steps. A first step is to form a mixture comprising a nucleic acid population, at least one blocked cleavable primer, a hybrid RNase H2 protein, dNTPs, DNA polymerase, and a buffer, such that a hybrid double helix is ​​formed between the nucleic acid population and at least one blocked cleavable primer in the mixture. A second step is to cleave at least one blocked cleavable primer with the hybrid RNase H2 protein to produce at least one active primer that can be extended by DNA polymerase. A third step is to extend at least one active primer with DNA polymerase in the buffer under conditions that allow amplification of one or more template nucleic acids from the nucleic acid population, thereby producing a unit replication sequence of the template nucleic acid. In the first related matter, the hybrid RNase H2 protein is selected from Q48R SEL29 (SEQ ID NO: 18) or other hybrid RNase H2 proteins. In the second related matter, DNA polymerase is high-fidelity archaeal DNA polymerase or other DNA polymerase. In the third related matter, buffer is high-fidelity archaeal DNA polymerase buffer.

[0083] In a tenth aspect, a method for performing massively parallel sequencing is provided. The method comprises several steps. The first step is to prepare a library population of template nucleic acids using a nucleic acid population, a hybrid RNase H2 mutant protein, at least one blocked cleavable primer, DNA polymerase, dNTPs and buffers in a PCR method. The second step is to sequence several desired template nucleic acids derived from the library population of template nucleic acids. In the first aspect, the hybrid RNase H2 protein is selected from Q48R SEL29 (SEQ ID NO: 18) or other hybrid RNase H2 proteins.

[0084] In an eleventh embodiment, a method is provided for detecting an SNP-containing nucleic acid template from a nucleic acid template amplification product library. The method comprises several steps. A first step is to form a mixture comprising: a nucleic acid template amplification product library; at least one blocked cleavable primer; a hybrid mutant RNase H2 protein; dNTPs; DNA polymerase; and a buffer. A hybrid duplex is formed between at least one blocked cleavable primer in the mixture and an SNP-containing nucleic acid template in the nucleic acid template amplification product library. A second step is to cleave at least one blocked cleavable primer of the hybrid duplex with the hybrid RNase H2 protein to create at least one active primer that can be extended by DNA polymerase on the hybrid duplex. A third step is to extend at least one active primer in the duplex with DNA polymerase in a buffer under conditions that allow amplification of one or more template nucleic acids from the nucleic acid template amplification product library, thereby detecting an SNP-containing nucleic acid template. In the first related matter, the method comprises a hybrid mutant RNase H2 protein selected from Q48R SEL29 (SEQ ID NO: 18) or other hybrid RNase H2 proteins. In the second related matter, the method comprises a buffer which is a high-fidelity archaeal DNA polymerase buffer.

[0085] In a twelfth embodiment, a method for carrying out a loop-mediated amplification reaction is provided. The method comprises two steps. The first step is to form a mixture comprising a nucleic acid template; four blocked cleavable primers forming a double helix with the nucleic acid template, which is a substrate for the RNase H2 protein; an RNase H2 protein selected from Q48R SEL29 (SEQ ID NO: 18) or other hybrid RNase H2 proteins; a DNA polymerase protein; dNTP; and a buffer. The second step is to carry out an isothermal amplification cycle with the mixture.

[0086] In a thirteenth embodiment, a method is provided for performing an rhPCR assay in which primer dimer formation is reduced. The method includes the step of performing primer extension with Q48R SEL29 RNase H2 (SEQ ID NO: 18). The reduction in primer dimer formation corresponds to a reduction in the amount of primer dimers formed during an rhPCR assay with Q48R SEL29 RNase H2 (SEQ ID NO: 18) compared to an rhPCR assay performed with wild-type PaRNase H2 (SEQ ID NO: 1).

[0087] In a fourteenth embodiment, a method is provided for performing an rhPCR assay with improved mapping and on-target rates for a desired product. The method includes the step of performing primer extension with Q48R SEL29 RNase H2 (SEQ ID NO: 18). The improvement in mapping and on-target rates corresponds to an increase in mapping and on-target amplification of the desired product formed during the rhPCR assay with Q48R SEL29 RNase H2 (SEQ ID NO: 18) compared to an rhPCR assay performed with wild-type PaRNase H2 (SEQ ID NO: 1).

[0088] Finally, the RNase H2 polypeptide of the present invention is suitable for use in a highly efficient amplification method, BaseX-based PCR amplification, which is disclosed in U.S. Patent Publication US10273534(B2), the contents of which are incorporated herein by reference in their entirety. [Examples]

[0089] The present invention is further illustrated by reference to the following embodiments. However, it should be noted that these examples, like the embodiments described above, are illustrative and should not be understood as limiting the permissible scope of the invention in any true way.

[0090] [Example 1] Generation of SEL28 hybrid RNase H2 protein and SEL29 hybrid RNase H2 protein via recombinant reshuffling Mutant RNase H2 proteins were synthesized using in vitro DNA recombination and directed molecular evolution. Under an agreement with Integrated DNA Technologies, Altravax® Inc. (Sunnyvale, CA) created a library of 5,500 mutants. Initial screening conducted at IDT selected SEL28 and SEL29 mutants, which exhibited increased mismatch recognition in the RNase H2 cleavage reaction. The mutants were created within a pET-27b(+) plasmid vector inside Escherichia coli (E. coli) BL21(DE3). Proteins expressed based on the T7 system contain a pelB signal sequence at the N-terminus, a mutant RNase H2 gene, a human herpes simplex virus 2 epitope tag, and a 6-histidine tag at the C-terminus. E. collie cells were grown in 12 mL of LB culture medium with 50 μg / mL kanamycin (Teknova®, Hollister, CA) using 50 mL of TPP TubeSpin® Bioreactors (Techno Plastic Products AG, Trasadingen, Switzerland). RNase H2 expression was induced over 20 hours at 37°C using an overnight Express® Autoinduction System 1 (MilliporeSigma®, Burlington, MA). The bacterial expression strain was grown with shaking at 250 rpm using a MaxQ® 4000 orbital shaker (ThermoFisher Scientific®, Grand Island, NY). The cells were centrifuged in a ThermoScientific Sorvall® Legend XTR centrifuge at 7,500 × g for 10 minutes, and the supernatant was discarded.The cell paste was stored at -80°C and resuspended in 0.6 mL of cell resuspension buffer containing 50 mM NaCl, 40 mM Tris-HCl pH 8.0, 2.5 mM MgCl2, 0.5 mM CaCl2, 1x BugBuster® Extraction Reagent (MilliporeSigma®, Burlington, MA), 1x cOmplete® EDTA-free protease inhibitor cocktail (MilliporeSigma®, Burlington, MA), 0.1 mg / mL lysozyme (approximately 600 units, ThermoFisher® Scientific, Grand Island, NY), and 4 U / mL Ambion® DNase I (ThermoFisher® Scientific, Grand Island, NY). Dissolution required 15 minutes at 25°C with shaking at 120 rpm. Insoluble substances were removed by centrifugation at 16,000 × g for 20 minutes. DNase I and native E. Cory protein were denatured at 75°C for 15 minutes. Insoluble denatured proteins were again removed by centrifugation at 16,000 × g for 15 minutes. For protein purification, the Capturem® His-Tagged Miniprep kit (Takara Bio®, Mountain View, CA) was used. The conjugated column from the kit was washed with cell resuspension buffer, and the supernatant containing RNase H2 was loaded onto the column. The solution was removed from the column by centrifugation at 11,000 × g for 1 minute. The column was washed twice with 200 μL of washing buffer (20 mM Na3PO4, 150 mM NaCl, pH 7.6) with 20 mM imidazole added.The RNase H2 enzyme was eluted with 200 μL of elution buffer (20 mM Na3PO4, 500 mM NaCl, 500 mM imidazole, pH 7.6) and dialyzed overnight with 1 L of double-concentration storage buffer F (pH 8.4, 40 mM Tris-HCl, 0.2 mM EDTA, 200 mM KCl) using a D-Tube® Dialyzer Midi, MWCO 6-8 kDa (MilliporeSigma®, Burlington, MA). The dialyzing buffer in the dialyzer was replaced at least once. The sample was recovered from dialyzing and mixed with a mixture of 99.8% (v / v) glycerol and 0.2% (v / v) Triton X-100 in a 1:1 volume ratio. These purified and concentrated RNase H2 solutions were stored at -20°C. The purity of these samples was estimated by SDS gel electrophoresis using Any kD® Mini-PROTEAN® TGX Stain-Free® Protein Gels (Bio-Rad®, Hercules, CA). The RNase H2 enzyme exhibited a major protein band (>75%) in all purified samples, and its position corresponded to a molecular weight of 28.9 kg per mole compared to Precision Plus Protein® Unstained Standards (Bio-Rad®, Hercules, CA).

[0091] The amino acid sequences of the mutant proteins are shown in Table 1, SEQ ID NOs. 89 and 90. Sequencing was performed using the Applied Biosystems BigDye Terminators v3.1 kit. Plasmid DNA was isolated from bacterial strains using the Wizard® Plus SV Minipreps DNA Purification System (Promega, Madison, WI) according to the manufacturer's protocol. Sequencing data were collected using the Applied Biosystems 3130 Genetic Analyzer.

[0092] [Example 2] The Q48R SDM mutant and the A107V SDM mutant within SEL29 RNase H2 increase the enzyme activity in Thermocox kodacarensis DNA polymerase reaction buffer compared to SEL29 RNase H2. The (His)6-tagged mutants, SEL28 RNase H2 and SEL29 RNase H2 proteins, were generated by site-directed mutagenesis (SDM) (see, for example, Weiner M. et al., Gene, 151:119-123 (1994)). The primers used for SDM of SEL28 RNase H2 and SEL29 RNase H2 are shown in Table 2, SEQ ID NOs. 4-13. The mutant sequences were validated, and the proteins were expressed in E. kohlii using standard methods. Purification was performed using charged Ni as previously described (Dobosy et al., 2011; and US Patent No. 8,911,948B2). 2+ Affinity purification on a column was performed. The amino acid sequences of the mutant proteins are shown in Table 3, SEQ ID NOs. 2-3, 14-20.

[0093] [Table 2]

[0094] [Table 3] TIFF0007900392000006.tif252163TIFF0007900392000007.tif76161

[0095] To determine by rhPCR whether P13S SEL28 RNase H2, A107V SEL28 RNase H2, and P13S / A107V SEL28 RNase H2 or P13S SEL29 RNase H2, Q48R SEL29 RNase H2, A107V SEL29 RNase H2 or P13S / A107V SEL29 RNase H2 increase the activity in KOD DNA polymerase reaction buffer compared to WT PaRNase H2, a quantitative rhPCR assay targeting the rs4939827 SNP in SMAD7 was designed. This SNP has already been used to characterize the efficiency and specificity of rhPCR (Dobosy et al., 2011; and U.S. Patent No. 8,911,948B2), and its response under differential conditions is well understood. The primers used in this assay are shown in Table 4, SEQ ID NOs. 21-23. The assay was performed in a reaction volume of 10 μL. Thermal cycling and data acquisition were performed on a CFX384® Real Time System (Bio-Rad®, Hercules, CA). In short, a 200 nM (2 picomoles) protected forward primer (SEQ ID NO: 22) and a 200 nM (2 picomoles) unblocked reverse primer (SEQ ID NO: 23), or a 200 nM (2 picomoles) unblocked forward primer (SEQ ID NO: 21) and a 200 nM (2 picomoles) unblocked reverse primer (SEQ ID NO: 23) were mixed with 2.5 mM (total concentration) MgSO4, 0.2 mM (each concentration) dNTP (MilliporeSigma®, Burlington, MA), and 0.5 times the concentration of EvaGreen® dye (Biotium Inc., Fremont, CA) in 1x internal KOD buffer (ROKStar buffer v2.0 or v1.66) (IDT, Coralville, IA).RNase H2 dilution buffer (IDT, Coralville, IA) or 21 femtomol of WT Pa (SEQ ID NO: 1), SEL28 (SEQ ID NO: 2) RNase H2 enzyme, SEL29 (SEQ ID NO: 3) RNase H2 enzyme, P13S SEL28 (SEQ ID NO: 14) RNase H2 enzyme, A107V SEL28 (SEQ ID NO: 15) RNase H2 enzyme, P13S / A107V SEL28 (SEQ ID NO: 16) RNase H2 enzyme, P13S SEL29 (SEQ ID NO: 17) RNase H2 enzyme, Q48R SEL29 (SEQ ID NO: 18) RNase H2 enzyme, A107V SEL29 (SEQ ID NO: 19) RNase H2 enzyme, or P13S / A107V SEL29 (SEQ ID NO: 20) RNase H2 enzyme was added to each reaction product. For the rs4939827 SNP, 10 ng of cell line genomic DNA (cell line NA12878; Coriell Institute for Medical Research, Camden, NJ) representing a homozygous genotype was added to each reactant. The reaction was carried out in three successive steps, and the results were averaged. The reaction was carried out under the following conditions: 95°C. 3:00 →(95℃ 0:10 →60℃ 0:30 The assay was cycled under 75°C. Fluorescence data for the inserted EvaGreen® was collected after each extension time point. After completing the assay, the data was analyzed and the automatic determination function of the Bio-Rad CFX Manager® software was used to determine the C q The values ​​were calculated. The results are shown in Table 5.

[0096] [Table 4]

[0097] [Table 5] TIFF0007900392000010.tif180162

[0098] These data show that Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 increased the enzyme activity in Thermococcus kodacarensis DNA polymerase reaction buffer compared to background SEL29 RNase H2, while P13S SEL29 RNase H2 and P13S / A107V SEL29 RNase H2 did not. In addition, P13S SEL28 RNase H2, A107V SEL28 RNase H2, and P13S / A107V SEL28 RNase H2 did not increase the enzyme activity in Thermococcus kodacarensis DNA polymerase reaction buffer compared to background SEL28 RNase H2. Without the addition of RNase H2, blocked primers are not cleaved and therefore cannot support PCR. The use of patented exonuclease-resistant blocked primers is necessary to prevent deprotection of the primers by high-fidelity DNA polymerases. While WT(Pa)RNase H2 can cleave blocked primers, it results in delayed amplification, leading to ΔC of 12.4 cycles in ROKstar buffer v2.0 and 21.1 cycles in ROKstar buffer v1.66 ​​compared to unblocked primers. q This results in ΔC for amplification delay. q The quantitative analysis of increases to 17.1 cycles for SEL28 RNase H2 and 24.6 cycles for SEL29 RNase H2 in ROKstar buffer v2.0, and to 30.6 cycles for SEL28 RNase H2 and 30.3 cycles for SEL29 RNase H2 in ROKstar buffer v1.66. The ΔC for amplification delay is also shown. qQuantification is decreased by Q48R SEL29 RNase H2 (17.2 cycles in ROKstar buffer v2.0 and 25.4 cycles in ROKstar buffer v1.66) and A107V SEL29 RNase H2 (13.9 cycles in ROKstar buffer v2.0 and 21.9 cycles in ROKstar buffer v1.66). P13S / A107V SEL29 RNase H2 increases the quantification of ΔC q for amplification delay to 26.9 cycles in ROKstar buffer v2.0 and 32.8 cycles in ROKstar buffer v1.66, while P13S SEL28 RNase H2 and P13S SEL29 RNase H2 have no apparent activity in ROKstar buffer v2.0 or ROKstar buffer v1.66. In addition, P13S / A107V SEL28 RNase H2 increases the quantification of ΔC q for amplification delay to 21.5 cycles in ROKstar buffer v2.0 and 31.8 cycles in ROKstar buffer v1.66. A107V SEL28 RNase H2 increases the quantification of ΔC q for amplification delay to 20.0 cycles in ROKstar buffer v2.0 and 35.8 cycles in ROKstar buffer v1.66. None of Q48R P.a. RNase H2, A107V P.a. RNase H2, and P13S / A107V P.a. RNase H2 increases the enzyme activity in Thermococcus kodakarensis DNA polymerase reaction buffer, and P13S P.a. RNase H2 also has a low but measurable enzyme activity in Thermococcus kodakarensis DNA polymerase reaction buffer, so these results are in contrast to the mutations within P.a. RNase H2. Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 improve the enzyme activity (although to different extents) when used with Thermococcus kodakarensis DNA polymerase reaction buffer.

[0099] [Example 3] When the mismatch is located opposite the RNA, Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 increase mismatch recognition compared to WT RNase H2. The specific activity of the enzyme was determined using a fluorescence-based reaction rate assay. Sequences for the DNA substrate are shown in Table 6, SEQ ID NOs. 24-25. The substrate is a DNA hairpin with RNA bases matched within the double-stranded region. The probe was conjugated with 6-FAM (6-carboxyfluorescein) at its 3' end and with Iowa Black® FQ (SEQ ID NO: 24) at its 5' end. In an intact hairpin probe, the fluorescence of 6-FAM is quenched by the Iowa Black® FQ portion. RNase H2 cleaves the 3' end of the 6-FAM-containing probe at the 5' end of the RNA base, releasing it. Therefore, the fluorescence of 6-FAM is no longer quenched and can fluoresce. A DNA hairpin containing RNA base but without a fluorophore or quencher was used as a competitor (SEQ ID NO: 25). The assay was performed in a reaction volume of 10 μL. Data acquisition was performed using a LightCycler® 480 II (Roche Life Science, Indianapolis, IN). Briefly, 1x concentration rhAmp® Backbone was used. v3 was used in combination with 200 nM (2 picomoles) labeled hairpin (SEQ ID NO: 24) and 10 μM (100 picomoles) competing hairpin (SEQ ID NO: 25). 0.5, 1.0, 2.0, or 5.0 femtomoles of WT PaRNase H2 (SEQ ID NO: 1) or mutant RNase H2 (SEQ ID NOs: 18-19) were added to each reactant. The reaction mixture was initially kept at 4°C to prevent substrate cleavage before starting the assay. Samples were prepared at 65°C. The fluorescence excitation wavelength was 483 nm; the fluorescence emission wavelength was 533 nm. Fluorescence intensity was collected every 13.75 seconds for 135 minutes. For each reaction mixture, the initial rate and rate per femtomole were calculated. For each mutant, the rate per femtomole was calculated for WT, which had already been determined to have specific activity of 17 units per μg of enzyme. The values ​​were normalized in relation to those for PaRNase H2.Q48R SEL29 RNase H2 has a specific activity of 46.03 units per 1 μg of enzyme, while A107V SEL29 RNase H2 has a specific activity of 7.77 units per 1 μg of enzyme.

[0100] [Table 6]

[0101] To fully determine whether these mutant RNase H2 enzymes improve mismatch recognition for mismatches directly opposite RNA bases, a synthetic quantitative rhPCR assay was used, as previously described (Dobosy et al., 2011; and U.S. Patent No. 8,911,948B2). This assay allows for direct comparison of the effect of each specific single-nucleotide mismatch with that of a perfect match. The primers used in this assay are shown in Table 7, SEQ ID NOs. 26-31. The assay was performed in a reaction volume of 10 μL. Thermal cycling and data acquisition were performed on a CFX384® Real Time System (Bio-Rad®, Hercules, CA). In short, 200 nM (2 picomoles) of blocked reverse primers (SEQ ID NOs. 28-31) and 200 nM (2 picomoles) of unblocked forward primers (SEQ ID NOs. 26), or 200 nM (2 picomoles) of unblocked reverse primers (SEQ ID NOs. 27) and 200 nM (2 picomoles) of unblocked forward primers (SEQ ID NOs. 26) were mixed with 1x iQ(trademark)SYBR(registered trademark)Green Supermix(registered trademark) (Bio-Rad(registered trademark), Hercules, CA). 5 mU of WT Pa(SEQ ID NOs. 1)RNase H2 enzyme, 40 mU of Q48R SEL29(SEQ ID NOs. 18)RNase H2 enzyme, or 5 mU of A107V SEL29(SEQ ID NOs. 19)RNase H2 enzyme were added to each reaction product. Each reactant was given 20,000 copies of their respective synthetic target sequences (SEQ ID NOs. 32-35), each with a different nucleotide directly opposite the RNA base. The reaction was carried out in triplicate, and the results were averaged. The reaction was performed under the following conditions: 95°C. 3:00 →(95℃ 0:10 →60℃ 0:30 The assay was cycled under 75 × ) conditions. Fluorescence data for the inserted SYBR® Green were collected after each extension time point. After the assay was completed, the data were analyzed, and for each corresponding combination, the mean C q Value and ΔC qThe values ​​were calculated. The results are shown in Tables 8 and 9.

[0102] [Table 7]

[0103] [Table 8] TIFF0007900392000014.tif140168TIFF0007900392000015.tif139167

[0104] [Table 9] TIFF0007900392000017.tif118168TIFF0007900392000018.tif135168

[0105] These data show a significant increase in mismatch recognition (approximately 14.0 cycles and 13.5 cycles, respectively) for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 when the mismatch is opposite rC. In particular, a large increase in mismatch recognition for rC:C pairs is observed for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 (21.9 cycles and 15.2 cycles, respectively). When the mismatch is opposite rU, a marked increase in mismatch recognition is observed for Q48R SEL29 RNase H2 (approximately 11.2 cycles), but no marked increase is observed for A107V SEL29 RNase H2 (approximately 4.3 cycles). When the mismatch is opposite rA, a significant change in mismatch recognition is observed for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 (approximately 8.5 cycles and 6.0 cycles, respectively). When the mismatch is opposite rG, the change in mismatch recognition is small; however, this lack of change in mismatch recognition is not significant, as mismatch recognition using WT RNase H2 was already quite effective for rG. These increases in mismatch recognition for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 are similar to the mismatch recognition for background SEL29 RNase H2. Both mutations improve mismatch recognition when the mismatch is located directly opposite the RNA base, but to different degrees and with different specificities.

[0106] [Example 4] When the mismatch is located on the 5' side of the RNA, Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 increase mismatch recognition compared to WT RNase H2. To determine the degree to which mismatches are identified when mismatches occur at the 5' end of the RNA nucleotide, an assay was designed targeting rs113488022 (a V600E SNP in the human BRAF gene) by positioning the SNP directly adjacent to the 5' end of the RNA, using the Q48R SEL29 RNase H2 enzyme and the A107V SEL29 RNase H2 enzyme. The primers used in this assay are shown in Table 10, SEQ ID NOs. 36-39. The assay was performed in a reaction volume of 10 μL. Thermal cycling and data acquisition were performed on a CFX384® Real Time System (Bio-Rad®, Hercules, CA). In short, a 200 nM (2 picomoles) blocked forward primer (SEQ ID NO: 38 or 39) and a 200 nM (2 picomoles) unblocked reverse primer (SEQ ID NO: 37), or a 200 nM (2 picomoles) unblocked forward primer (SEQ ID NO: 36) and a 200 nM (2 picomoles) unblocked reverse primer (SEQ ID NO: 37), were mixed into a 1x concentration of iQ(trademark)SYBR(registered trademark)Green Supermix(registered trademark). 5 mU or 10 mU of WT(SEQ ID NO: 1) PaRNase H2 enzyme, 5 mU or 10 mU of SEL29(SEQ ID NO: 3) RNase H2 enzyme, 20 mU or 40 mU of Q48R SEL29(SEQ ID NO: 18) RNase H2 enzyme, or 5 mU or 10 mU of A107V SEL29(SEQ ID NO: 19) RNase H2 enzyme were added to each reaction product. Each reactant was given 2,000 copies of a synthetic double-stranded gBlock® (IDT, Coralville, IA) template corresponding to the homozygous genotype of the rs113488022 SNP (SEQ ID NO: 40 or 41). The reaction was carried out in triplicate. The reaction was performed under the following conditions: 95°C. 3:00 →(95℃ 0:10 →60℃ 0:30 The assay was cycled under 65 × 5. Fluorescence data for the inserted SYBR® Green was collected after each extension time point. After the assay was completed, the data was analyzed, and for each corresponding combination, the mean Cq Value and ΔC q The values ​​were calculated. The results are shown in Table 11.

[0107] [Table 10]

[0108] [Table 11] TIFF0007900392000021.tif99164

[0109] These data demonstrate that Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 significantly improve mismatch recognition at the 5' end of RNA nucleotides. ΔC for TrG primers. q The quantification of increased from 3.8 cycles with WT PaRNase H2 to 5.9 cycles with A107V SEL29 RNase H2 (for 5mU of WT RNase H2 and A107V SEL29 RNase H2), and from 4.5 cycles with WT PaRNase H2 to 6.9 cycles with Q48R SEL29 RNase H2 (for 5mU of WT RNase H2 and 40mU of Q48R SEL29 RNase H2). For ArG primers, ΔC q The quantification increased from 10.1 cycles with WT PaRNase H2 to 11.2 cycles with A107V SEL29 RNase H2 (for 5mU of WT RNase H2 and A107V SEL29 RNase H2), but decreased from 10.9 cycles with WT PaRNase H2 to 10.2 cycles with Q48R SEL29 RNase H2 (for 5mU of WT RNase H2 and 40mU of Q48R SEL29 RNase H2). ΔC when using WT enzyme qHowever, since this primer was already quite effective, the change in mismatch recognition for the ArG primer is not very significant. These increases in mismatch recognition for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 are similar to the mismatch recognition for background SEL29 RNase H2. Therefore, Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 improve mismatch recognition when the mismatch is located on the 5' side of the RNA base.

[0110] [Example 5] When the mismatch is located on the 3' side of the RNA, Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 increase mismatch recognition compared to WT RNase H2. To determine whether the Q48R SEL29 RNase H2 enzyme and the A107V SEL29 RNase H2 enzyme can improve mismatch recognition when the mismatch is positioned at the 3' end of the RNA nucleotide, we designed an assay targeting rs7583169 SNP and rs3117947 SNP with the mismatch positioned directly adjacent to the 3' end of the RNA. The primers used in this assay are shown in Table 12, SEQ ID NOs. 42-47. The assay was performed in a reaction volume of 10 μL. Thermal cycling and data acquisition were performed on a CFX384® Real Time System (Bio-Rad®, Hercules, CA). In short, a 200 nM (2 picomoles) blocked forward primer (SEQ ID NO: 43 or 46) and a 200 nM (2 picomoles) unblocked reverse primer (SEQ ID NO: 44 or 47), or a 200 nM (2 picomoles) unblocked forward primer (SEQ ID NO: 42 or 45) and a 200 nM (2 picomoles) unblocked reverse primer (SEQ ID NO: 44 or 47) were mixed with 3.0 mM (total concentration) MgCl2 and 0.5 times the concentration of EvaGreen® dye (Biotium Inc., Fremont, CA) in a 1x concentration of PrimeTime® Gene Expression Master Mix (IDT, Coralville, IA). 5 mU or 10 mU of WT (SEQ ID NO: 1) PaRNase H2 enzyme, 50 mU or 100 mU of Q48R SEL29 (SEQ ID NO: 18) RNase H2 enzyme, or 20 mU or 40 mU of A107V SEL29 (SEQ ID NO: 19) RNase H2 enzyme was added to each reactant. 20 ng of cell line genomic DNA (cell lines NA12878 and NA24385; Coriell Institute for Medical Research, Camden, NJ) representing two homozygous genotypes at rs7583169 SNP and rs3117947 SNP was added to each reactant. The reaction was carried out in triplicate, and the results were averaged. The reaction was carried out under the following conditions: 95°C 3:00 →(95℃0:10 →60℃ 0:30 The assay was cycled under 65 × 5°C conditions. Fluorescence data for the inserted EvaGreen® were collected after each extension time point. After the assay was complete, the data were analyzed, and for each corresponding combination, the mean C q Value and ΔC q The values ​​were calculated. The results are shown in Table 13.

[0111] [Table 12]

[0112] [Table 13]

[0113] These data show that Q48R SEL29 and A107V SEL29 significantly increase mismatch recognition at the 3' end of RNA nucleotides. (ΔC for mismatch recognition of rs7583169 SNP) q The quantification increased from 0.0 cycles with WT PaRNase H2 to 1.4 cycles with A107V SEL29 RNase H2 (for 5mU of WT RNase H2 and 40mU of A107V SEL29 RNase H2), and from 0.0 cycles with WT PaRNase H2 to 11.5 cycles with Q48R SEL29 RNase H2 (for 5mU of WT RNase H2 and 100mU of Q48R SEL29 RNase H2). rs3117947 ΔC for SNP mismatch identification qThe quantification increased from 10.7 cycles with WT PaRNase H2 to 22.3 cycles with A107V SEL29 RNase H2 (for 5mU of WT RNase H2 and 40mU of A107V SEL29 RNase H2), and from 10.7 cycles with WT PaRNase H2 to 20.4 cycles with Q48R SEL29 RNase H2 (for 5mU of WT RNase H2 and 100mU of Q48R SEL29 RNase H2). These increases in mismatch identification for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 are significantly greater than the mismatch identification for background SEL29 RNase H2. Therefore, Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 improve mismatch recognition when the mismatch is located on the 3' side of the RNA base.

[0114] [Example 6] Use of Q48R SEL29 hybrid RNase H2 protein and A107V SEL29 hybrid RNase H2 protein in the LAMP reaction This example outlines a method supporting the use of hybrid RNase H2 proteins to reduce primer dimer formation in the LAMP reaction.

[0115] To assess the functionality of rhPrimers for LAMP protocols containing different RNase H2 proteins (WT RNase H2 protein or hybrid RNase H2 protein), three assays could be designed using (1) an unmodified control primer; "Gen1," which is an rDDDDMx primer [in the sequence, "r" is an RNA base, "D" is a DNA base, "m" is a mismatch, and "x" is a C3 spacer]; and "Gen2," which is an rDxxDM primer. Details of the assays used are described in Tables 14, 15, and 16. LAMP reaction mixtures utilizing each type of primer are held at 25°C (room temperature) for 0 or 2 hours prior to testing. This allows for the formation of primer dimer products. After holding at room temperature, all reactions are carried out in a BioRad CFX384 or Roche LightCycler 480 at 65°C for 2 hours. The generation of signals in all of these reaction solutions is carried out by adding 1x concentration of EvaGreen to the reaction (see Biotechnology Letters, December 2007, Vol. 29, No. 12, pp. 1939-1946).

[0116] [Table 14]

[0117] [Table 15] TIFF0007900392000026.tif116164

[0118] [Table 16] TIFF0007900392000028.tif65164

[0119] Each assay may be performed as follows: The sample is Coriell gDNA or lambdafage genomic DNA. Each assay condition may be tested in triplicate with a sample input of 5 ng lambdafage genomic DNA or 20 ng human genomic DNA. For each assay, the reaction may be tested using unmodified primer insertion dye (e.g., EvaGreen) and cleavable blocked primers with insertion dye. For each assay, the reaction may be tested using zero-level or titration-level RNase H2 (WT RNase H2 protein or hybrid RNase H2 protein).

[0120] Each assay is tested in triplicate. Comparison between the unmodified LAMP assay and the modified LAMP assay is performed by comparing the length of time required for the formation of signaling products. Hybrid RNase H2 proteins in the LAMP reaction mixture are expected to yield these products with a significantly delayed response compared to reaction mixtures containing conventional wild-type RNase H2 proteins.

[0121] 25 μL of EvaGreen reaction mixture 12.5 μL of 2x concentration Master Mix (25°C, pH 8.8, 1x concentration 20 mM Tris, 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, 0.01% Tween-20, 1.4 mM dNTPs) 1.6 μM FIP primer 1.6 μM BIP primer 1x concentration EvaGreen dye 0.2 μM F3 primer 0.2 μM B3 primer 8U BST DNA polymerase (New England Biolabs: https: / / www.neb.com / product / m0275-bst-dna-polymerase-large-fragment) 1 μL of hybrid RNase H2 protein (buffer D is not used as an RNase H2 control) Nuclease-free water up to 25 μL 2 μL of sample (10 ng / μL human gDNA or 2.5 ng / μL lambda genomic DNA) Includes.

[0122] [Example 7] Q48R SEL29 RNase H2 improves the quality of NGS libraries compared to wild-type PaRNase H2 in a multiplex rhPCR unit replication sequence sequencing workflow using a 177plex assay panel. A modified rhAmpSeq protocol was adapted for the detection of low-frequency mutants. This protocol uses both a high-fidelity DNA polymerase to reduce amplification errors and a UMI (unique molecular identifier) ​​to correct errors. Q48R SEL29 RNase H2 was compared to wild-type PaRNase H2 in this system to determine whether dimerization was reduced.

[0123] Following each step, two PCR cycling steps are performed, accompanied by SPRI (Solid Phase Reversible Immobilization, Beckman Coulter Life Sciences, Indianapolis, IN) cleanup. The objective of the first PCR step (PCR1) is to incorporate a 6-nucleotide degenerate UMI on each side of the target unit replica sequence. This step also involves the use of 3'-blocked primers, requiring RNase H2 to cleave the protective agent, allowing high-fidelity DNA polymerase to extend and amplify each target. The target-specific assay panel contains 177 patented primer pairs, resulting in approximately 20% of all reads becoming primer dimers when wild-type PaRNase H2 is present in PCR1. Because this panel yields a large number of primer dimers, it is ideal for investigating Q48R SEL29 RNase H2.

[0124] Various 10-fold titrations of Q48R SEL29 RNase H2 and wild-type PaRNase H2 (87.5 mU / uL, 175 mU / uL, and 350 mU / uL) were prepared in RNase H2 storage buffer (Integrated DNA Technologies, Coralville, IA). Each PCR1 reaction mixture had a final volume of 20 μL and contained 10 nM (200 femtomoles) of each forward and reverse primer, 0.03 U / μL of Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific, Waltham, MA), 20 ng of cell line genomic DNA (cell line, NA24385; Coriell Institute for Medical Research, Camden, NJ), and RNase H2 in a patented high-fidelity polymerase buffer with a final concentration of 1x. Thermal cycling was performed on a T100 Thermal Cycler (Bio-Rad®, Hercules, CA) under the cycling conditions listed in Table 17. Following PCR1 thermal cycling, SPRI cleanup was performed promptly. 1.25 times (25 μL) of AMPure magnetic beads (Beckman Coulter Life Sciences, Indianapolis, IN) were added to each well and mixed thoroughly. The plate was incubated on a workbench at room temperature for 5 minutes, followed by incubation on a magnet for 5 minutes. The supernatant was discarded, and the library was washed twice with 80% ethanol. The sample was eluted in 22 μL of IDTE pH 7.5 (IDT, Coralville, IA). 20 μL of the eluted product was fed as input for PCR2.

[0125] [Table 17]

[0126] The purpose of the second PCR step (PCR2) was to amplify the PCR1 product and add UMI (unique molecular identifiers) sequences for pooling and sequencing purposes. PCR2 does not require the use of RNase H2. The PCR2 reaction was performed in a final volume of 50 μL. To achieve a 1x final concentration, a 2x concentration of the high-fidelity polymerase buffer used in PCR1 was added to each eluted sample from PCR1. Each reaction mixture also had a unique combination of i5 hAmpSeq index primer (IDT) and i7r IDT, each at 500 nM. Thermal cycling was performed on a T100 Thermal Cycler (Bio-Rad®, Hercules, CA) under the cycling conditions listed in Table 18. SPRI cleanup was performed immediately after PCR2, which was identical to the method listed above, except for the AMPure bead concentration used at 0.9x after PCR2. The sample was eluted in 22 μL of IDTE pH 7.5 (IDT, Coralville, IA). 20 μL of the eluted product was taken and stored at -20°C until sequencing.

[0127] [Table 18]

[0128] The resulting library was pooled in equivolume (5 μL) for sequencing. This pool was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher) and diluted to a final concentration of 4 nM. Equivolute library pools were combined with 0.2 N NaOH to denature the libraries. This reaction mixture was then diluted to a final concentration of 8 pM, containing 2.5% PhiX (Illumina, San Diego, CA) spike-in. This reaction mixture was loaded into a MiSeq 300 cycle kit (Illumina, San Diego, CA) and tested (cluster density: 1002 ± 34; Q30: 93.13%). The results were processed through our proprietary IDT rhAmpSeq VII bioinformatics analysis pipeline.

[0129] For the purposes of this disclosure, the calculations and terms relating to the following results are hereby listed. The amount of primer dimers obtained during sequencing library amplification is defined by the dimer ratio. The rhAmpSeq VII pipeline calculates this by taking the total count of identified dimers and dividing by the number of QC-passing reads (total number of reads that passed the chastity filter (cf)). The mapping ratio is calculated by taking the total number of mapped reads and dividing by the number of QC-passing reads. The on-target ratio is calculated by taking the total number of on-target reads and dividing by the number of QC-passing reads. Total unit replication uniformity or ≥0.2x unit replication uniformity is calculated as the percentage of normal unit replication sequences that are greater than or equal to 0.2x the mean coverage of unit replication sequences. The dropout ratio or ≤0.05x uniformity is calculated as the percentage of normal unit replication sequences that are less than 0.05x the mean coverage of unit replication sequences. The uniformity distribution of unit replicate sequences is plotted within the following ranges for a given sample: 0.1–0.2x, 0.2–0.5x, 0.5–1.5x, 1.5–2.5x, and 2.5–5x, comparing the coverage of each unit replicate sequence with the mean unit replicate sequence coverage.

[0130] The rhAmpSeq VII pipeline also identifies the primers that contribute to the formation of each primer dimer. The normalized dimer count reflects the percentage contribution of each primer dimer to the total dimer percentage, and is expressed by the following formula:

[0131]

number

[0132] For all test concentrations, Q48R SEL29 RNase H2 reduced the dimerization rate compared to wild-type PaRNase H2, and also increased the mapping and on-target rates. At the lowest concentration (8.75 mU / μL), Q48R SEL29 RNase H2 yielded a library with 4% dimerization compared to the control wild-type PaRNase H2, which yielded 11% dimerization (Figure 1, Panel A).

[0133] The mapping and on-target rates for libraries prepared with the lowest concentration of Q48R SEL29 RNase H2 (8.75 mU / μL) increased to 96% compared to 88% for wild-type PaRNase H2 (Figure 1, panels B and C).

[0134] The dimer ratio is divided into individual primer dimers and then normalized to determine the contribution of each primer dimer to the total dimer ratio as described above (normalized dimer count). Q48R SEL29 RNase H2 reduces the amount of the largest primer dimer by half. For example, in a library prepared with the lowest concentration of Q48R Mut 29 RNase H2 (8.75 mU / μL), the top dimer identified in all sample libraries (NOTCH1_19.GAP95.9623.relax.t0_FOR:NOTCH1_9.GAP104.1246.relax.t0_REV) accounts for only 1.23% of the total dimer ratio, compared to 2.83% for wild-type PaRNase H2. In the case of the Q48R SEL29 RNase H2 library, some dimers are reduced by more than half, resulting in 0.11% dimers, which is 75% less than wild-type PaRNase H2. In this case, these dimers contribute 0.47% to the total dimer ratio (Table 19 and Figure 2).

[0135] [Table 19] TIFF0007900392000033.tif225165TIFF0007900392000034.tif57161

[0136] The ability of Q48R SEL29 RNase H2 to reduce dimerization rate does not affect metrics of library yield, overall homogeneity, and dropout rate or homogeneity distribution. Q48R SEL29 RNase H2 yields similar library yields compared to wild-type PaRNase H2 enzyme at all titration concentrations, as supported by mean unit replication sequence coverage (Figure 3, Panel A). In addition, overall unit replication sequence homogeneity (≥0.2x) and unit replication sequence dropout rate (≤0.05x) are comparable between Q48R SEL29 RNase H2 and wild-type Pa enzyme at all titration concentrations (Figure 3, Panels B and C). Furthermore, the homogeneity distribution between Q48R SEL29 and wild-type PaRNase H2 is also considered to be similar (Figure 4). Q48R SEL29 RNase H2 reduces primer dimerization during library preparation in the rhAmpseq workflow compared to standard wild-type PaRNase H2, without altering other important sequencing metrics.

[0137] In summary, these data demonstrate that Q48R SEL29 RNase H2 improves the creation of multiplex next-generation sequencing libraries using high-fidelity DNA polymerase in high-fidelity buffer.

[0138] [Example 8] Exemplary amino acid sequence and nucleic acid sequence encoding RNase H2 protein Exemplary amino acid and nucleic acid sequences encoding the RNase H2 protein are shown below.

[0139] [Table 20] TIFF0007900392000036.tif250161TIFF0007900392000037.tif251161TIFF00079003920 00038.tif249161TIFF0007900392000039.tif250161TIFF0007900392000040.tif145161

[0140] [Table 21] TIFF0007900392000042.tif232162

[0141] [Table 22] TIFF0007900392000044.tif250162TIFF0007900392000045.tif249161TIFF0007900392000046.tif105161

[0142] Cited references Joseph R Dobosy, Scott D Rose, Kristin R Beltz, Susan M Rupp, Kristy M Powers, Mark A Behlke and Joseph A Walder, RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers, BMC Biotechnology (2011), 11:80 Ayumu Muroya, Daisuke Tsuchiya, Momoyo Ishikawa, Mitsuru Haruki, Masaaki Morikawa, Shigenori Kanaya, and Kosuke Morikawa, Catalytic center of an archaeal type 2 ribonuclease H as revealed by X‐ray crystallographic and mutational analyses, Protein Science (2001), 10:707–714 Monika P. Rychlik, Hyongi Chon, Susana M. Cerritelli, Paulina Klimek, Robert J. Crouch, and Marcin Nowotny, Crystal Structures of RNase H2 in Complex with Nucleic Acid Reveal the Mechanism of RNA-DNA Junction Recognition and Cleavage, Molecular Cell(2010), 40:658~670

[0143] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as they are individually and specifically indicated to be incorporated herein by reference.

[0144] This specification describes preferred embodiments of the Invention, including the best way to carry out the Invention, which is known to the inventors. Variations of these preferred embodiments will be apparent to those skilled in the art by reading the foregoing. The inventors expect that those skilled in the art will utilize such variations as needed, and the inventors intend that the Invention may be carried out in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents thereof to the subject matter enumerated in the claims accompanying this Specified, as permitted by applicable law. Furthermore, unless otherwise indicated herein or unless the context clearly indicates otherwise, any combination of these possible variations of the elements described above is also encompassed in the Invention.

Claims

1. A hybrid RNase H2 protein comprising an amino acid sequence, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 14-20.

2. Recombinant nucleic acid encoding the hybrid RNase H2 protein according to claim 1.

3. A method for performing primer extension, comprising the step of contacting the hybrid RNase H2 protein described in claim 1 with a primer, a polynucleotide template, a nucleoside triphosphate, and a DNA polymerase under conditions suitable for a primer extension method, thereby producing an extension primer.

4. The method according to claim 3, wherein the DNA polymerase comprises a high-fidelity archaeal DNA polymerase, and the primer comprises a blocked cleavable primer having a cleavage domain and a blocking group that is bound to the 5' end of the 3' terminal residue of the blocked cleavable primer and inhibits the primer from being used as a template for DNA synthesis, and the primer extension method comprises performing a polymerase chain reaction (PCR) using the hybrid RNase H2 protein described in claim 1, and improving the 3'-side mismatch identification in primer extension in the primer:polynucleotide hybrid formed between the unblocked primer and the polynucleotide template, compared to mismatch identification in primer extension by performing PCR using the wild-type P. a. RNase H2 of SEQ ID NO:

1.

5. A reaction mixture comprising the hybrid RNase H2 protein according to claim 1, at least one primer including a blocked cleavable primer, a polynucleotide template, a nucleoside triphosphate, and a high-fidelity archaeal DNA polymerase.

6. A method for performing rhPCR, comprising the step of performing primer extension with the hybrid RNase H2 protein described in claim 1, a high fidelity archaeal DNA polymerase, and a blocked cleavable primer having a cleavage domain and a blocking group bound to the 5' end of the 3' terminal residue of the blocked cleavable primer and inhibiting the use of the primer as a template for DNA synthesis, wherein the hybrid RNase H2 protein described in claim 1 can be reversibly inactivated by chemical modification, an aptamer, or a blocking antibody.

7. The method according to claim 6, wherein the blocking group is selected from the group consisting of: one or more debase residues, or one member selected from the group consisting of RDDDDx, RDDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD [in the sequence, R is an RNA residue, D is a DNA residue, M is a mismatch residue, and x is a C3 spacer or another chemical group that is resistant to degradation and blocks extension by DNA polymerase].

8. The method according to claim 6, wherein the blocking group includes a label that enables detection of an extension amplification reaction, the label is bound to a blocked cleavable primer 3' from the cleavage site, and the label is a fluorophore or a mass tag.

9. The cleavage domain of a blocked cleavable primer can cleave the following: DNA residues, single RNA residues, two adjacent RNA residues, sequences of three or more RNA residues, debased residues, one or more 2' modified nucleosides, or internucleotide modified phosphate links. The method according to claim 6, comprising one or more of the above.

10. The method according to claim 6, wherein the cleavage domain comprises one or more 2'-modified nucleosides selected from the group consisting of 2'-O-alkylRNA nucleosides, 2'-O-methylRNA nucleosides, 2'-fluoronucleosides, locked nucleic acids, 2'-ethylene nucleic acid residues, 2'-alkyl nucleosides, 2'-aminonucleosides, and 2'-thionucleosides.

11. A method for amplifying a target DNA sequence, (a)(i) A blocked cleavable primer having a cleavage domain cleavable by the hybrid RNase H2 protein according to claim 1, wherein the blocking group is located on the 5' side of a blocking group attached to the 5' side of the 3' terminal residue of the blocked cleavable primer, the blocking group inhibits primer elongation and / or prevents the blocked cleavable primer from being used as a template for DNA synthesis; (ii) Sample nucleic acids that may or may not be target sequences; (iii) High fidelity archaeal DNA polymerase; and (iv) The hybrid RNase H2 protein according to claim 1, which can be reversibly inactivated by chemical modification, aptamer, or blocking antibody. A step of preparing a reaction mixture containing the following: (b) A step of hybridizing a blocked cleavable primer with a target DNA sequence to form a double-stranded substrate; (c) The step of removing the blocking group from the blocked cleavable primer by cleaving the hybridized blocked cleavable primer at a cleavage site within the cleavage domain or at a cleavage site adjacent to the cleavage domain using the hybrid RNase H2 protein described in claim 1. A method that includes this.

12. The method according to claim 11, wherein the blocking group is selected from the group consisting of: one or more debasement residues and one member selected from the group consisting of RDDDDx, RDDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD [in the sequence, R is an RNA residue, D is a DNA residue, M is a mismatch residue, and x is a C3 spacer or another chemical group that is resistant to degradation and blocks extension by DNA polymerase].

13. The method according to claim 11, wherein the blocking group includes a label that enables detection of an extension amplification reaction, the label is bound to a blocked cleavable primer 3' from the cleavage site, and the label is a fluorophore or a mass tag.

14. The method according to claim 11, wherein the cleavage domain comprises one or more of the following: a DNA residue, a single RNA residue, two adjacent RNA residues, a sequence of three or more RNA residues, a debased residue, one or more 2' modified nucleosides, or internucleotide modified phosphate links.

15. The method according to claim 14, wherein one or more 2'-modified nucleosides are selected from the group consisting of 2'-O-alkylRNA nucleosides, 2'-fluoronucleosides, locked nucleic acids, 2'-ethylene nucleic acid residues, 2'-alkyl nucleosides, 2'-aminonucleosides, and 2'-thionucleosides.

16. A kit for preparing extension primers, comprising at least one container providing the hybrid RNase H2 protein described in claim 1, and one or more further containers selected from the group consisting of (a) a container providing a primer capable of hybridizing with a predetermined polynucleotide template under primer extension conditions; (b) a container containing one or more blocked cleavable primers having a cleavage domain and a blocking group bound to the 5' end of the 3' terminal residue of the blocked cleavable primer and inhibiting the use of the primer as a template for DNA synthesis; (c) a container providing a nucleoside triphosphate; (d) a container providing a buffer suitable for primer extension; and (e) a container providing a high-fidelity archaeal DNA polymerase.

17. A kit for amplifying a target DNA sequence, comprising a reaction buffer containing the hybrid RNase H2 protein described in claim 1 and a high-fidelity archaeal DNA polymerase.

18. The kit according to claim 17, further comprising one or more oligonucleotide primers, the kit comprising a blocked cleavable primer having a cleavage domain and a blocking group that can be cleaved by the hybrid RNase H2 protein according to claim 1, wherein at least one oligonucleotide primer is located at the 5' end of the blocked cleavable primer or near the 3' end of the blocked cleavable primer and a blocking group, the blocking group inhibiting primer elongation and / or preventing the oligonucleotide primer from functioning as a template for DNA synthesis.

19. The kit according to claim 18, wherein the blocking group comprises one member selected from the group consisting of RDDDDx, RDDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD [in the sequence, R is an RNA residue, D is a DNA residue, M is a mismatch residue, and x is a C3 spacer or another chemical group that is resistant to degradation and blocks extension by DNA polymerase].

20. A method for preparing a library of amplification products of a template nucleic acid, A step of forming a mixture, wherein the mixture is Nucleic acid population; A blocked cleavable primer having a cleavage domain and a blocking group that is bound to the 5' end of the 3' terminal residue of the blocked cleavable primer and inhibits the primer from being used as a template for DNA synthesis; The hybrid RNase H2 protein of SEQ ID NO: 18; dNTP; High fidelity archaeal DNA polymerase; and High-fidelity archaeal DNA polymerase buffer Includes, A hybrid double helix is ​​formed between at least one blocked cleavable primer in the mixture and a population of nucleic acids; A step of cleaving at least one blocked cleavable primer with the hybrid RNase H2 protein of Sequence ID No. 18 to produce at least one active primer that can be extended by DNA polymerase; and In a buffer solution, under conditions that allow amplification of one or more template nucleic acids from a nucleic acid population, at least one active primer is extended by DNA polymerase, thereby producing an amplified product of the template nucleic acid. A method that includes this.

21. A method for performing massive parallel sequencing, Nucleic acid population; The hybrid RNase H2 protein of SEQ ID NO: 18; A blocked cleavable primer having a cleavage domain and a blocking group that is bound to the 5' end of the 3' terminal residue of the blocked cleavable primer and inhibits the primer from being used as a template for DNA synthesis; DNA polymerase; dNTP; and buffer solution A step of preparing a library population of template nucleic acids, comprising the step of performing PCR with a mixture containing, ;and Steps to sequence multiple desired template nucleic acids derived from a library of template nucleic acids. A method that includes this.

22. A method for detecting SNP-containing nucleic acid templates from a library of nucleic acid template amplification products, A step of forming a mixture, wherein the mixture is Nucleic acid template amplification product library; A blocked cleavable primer having a cleavage domain and a blocking group that is bound to the 5' end of the 3' terminal residue of the blocked cleavable primer and inhibits the primer from being used as a template for DNA synthesis; The hybrid RNase H2 protein of SEQ ID NO: 18; dNTP; DNA polymerase; and buffer solution Includes, A hybrid double helix is ​​formed between at least one blocked cleavable primer in the mixture and an SNP-containing nucleic acid template in a library of nucleic acid template amplification products; A step of cleaving at least one blocked cleavable primer of a hybrid double helix with the hybrid RNase H2 protein of SEQ ID NO: 18 to produce at least one active primer capable of extending the hybrid double helix by DNA polymerase; and In a buffer solution, under conditions that allow amplification of one or more template nucleic acids from a library of nucleic acid template amplification products, DNA polymerase is used to extend at least one active primer within a hybrid double helix, thereby enabling detection of SNP-containing nucleic acid templates. A method that includes this.

23. A method for carrying out a loop-mediated amplification reaction, A step of forming a mixture, wherein the mixture is Nucleic acid template; Four blocked cleavable primers, each having a cleavage domain and a blocking group bound to the 5' end of the 3' terminal residue of the blocked cleavable primer, which inhibits the primer from being used as a template for DNA synthesis, and which form a double helix together with a nucleic acid template that is a substrate for the RNase H2 protein; The hybrid RNase H2 protein of SEQ ID NO: 18; DNA polymerase protein; dNTP; and buffer solution Steps including; and The step of performing an isothermal amplification cycle using the mixture. A method that includes this.

24. A mixed product prepared using the method according to any one of claims 3, 6, 11, 20, 21, 22, and 23, wherein the mixed product prepared from the hybrid RNase H2 protein of SEQ ID NO: 18 contains a population with reduced primer dimer molecular species compared to the mixed product prepared with wild-type P. a. RNase H2 of SEQ ID NO:

1.

25. A method for performing an rhPCR assay in which primer dimer formation is reduced, comprising the step of performing primer extension with the hybrid RNase H2 protein of SEQ ID NO: 18, wherein the reduction in primer dimer formation corresponds to a reduction in the amount of primer dimers formed during an rhPCR assay using the hybrid RNase H2 protein of SEQ ID NO: 18 compared to an rhPCR assay performed using the wild-type P. a. RNase H2 of SEQ ID NO:

1.

26. A method for performing an rhPCR assay with improved mapping and on-target rates for a desired product, comprising the step of performing primer extension with the hybrid RNase H2 protein of SEQ ID NO: 18, wherein the improvement in mapping and on-target rates corresponds to an increase in mapping and on-target amplification of the desired product formed during the rhPCR assay with the hybrid RNase H2 protein of SEQ ID NO: 18, compared to an rhPCR assay performed with the wild-type P. a. RNase H2 of SEQ ID NO: 1.

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