RNase H2 mutants reduce primer dimers and off-target amplification in rhPCR-based amplicon sequencing with 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 under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 130,548, filed December 24, 2020, and entitled "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 November 9, 2021, and entitled "RNASE H2 MUTANTS THAT REDUCE PRIMER DIMERS AND OFF-TARGET AMPLIFICATION IN RHPCR-BASED AMPLICON SEQUENCING WITH HIGH-FIDELITY DNA POLYMERASES," the contents of which are incorporated by reference herein in their entireties.

[0002] FIELD OF THE PRESENT APPLICATION The present invention relates to type II RNase H (hereinafter referred to as RNase H2) hybrid enzyme variants and methods for cleaving nucleic acid strands to initiate, support, monitor or perform biological assays. [Background technology]

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

[0004] [ka]

[0005] RNase H2 enzyme isolated from the hyperthermophilic bacterium Pyrococcus abyssi (Pa) cleaves at the 5' end of ribonucleotides embedded in nucleic acid strands made from all other DNA. RNase H2-dependent PCR (rhPCR) improves PCR specificity by using a thermostable RNase H2 enzyme to remove the blocking group on the 3' end and cleave primers containing a single ribonucleotide near the 3' end (Dobosy et al., 2011; U.S. Patent No. 8,911,948 B2). The primer is initially unable to be extended by DNA polymerase, but can be extended once the blocking group is removed from the 3' end by RNase H2. Pa RNase H2 is sensitive to single base mismatches near ribonucleotides in DNA-RNA heteroduplexes and cleaves mismatch-containing templates at a much lower rate. This allows for preferential cleavage and extension of perfectly matched duplexes, resulting in increased specificity in rhPCR reactions and reducing primer-dimer formation and other off-target amplification.

[0006] Despite the enhancements it provides to PCR specificity, rhPCR currently has limitations. Apparent mismatch discrimination by rhPCR is lower than should be theoretically achievable. WT PaRNase H2 highly recognizes single-base mismatches directly opposite the RNA base, but the efficiency varies depending on the nature of the mismatch. In addition, the native enzyme has relatively limited mismatch discrimination at positions immediately 5' or 3' to the RNA base. Despite this limitation, placement of mutations at these positions can be advantageous. By way of example, a mismatch immediately 5' to the RNA can be used as a secondary selection step with the use of a discriminatory DNA polymerase (such as H784Q Thermus aquaticus DNA polymerase) in PCR amplification after primer cleavage (see U.S. Patent Application Serial No. 15 / 361280). Because mismatch recognition occurs every cycle rather than once during template conversion, placement of a mismatch at the 3' side of the RNA reduces the likelihood of template conversion.

[0007] Despite the usefulness of rhPCR, mismatch discrimination by wild-type PaRNase H2 results in some amplification of primer dimers as a possible "leakage" result. Primer dimers resulting from amplification of sequencing libraries are problematic because they bind to Illumina flow cells and undergo sequencing, but do not yield meaningful data. High levels of primer dimers reduce the fraction of reads that map to the target of interest, ultimately reducing assay sensitivity or requiring a significant increase in sequencing costs to generate the necessary number of on-target reads for detection of low-frequency variants. Detection of low-frequency variants may also be affected by the introduction of amplification errors during PCR. The use of high-fidelity DNA polymerases and optimized buffer conditions can reduce error rates. However, these buffer conditions reduce the enzymatic activity and mismatch sensitivity of wild-type PaRNase H2 in rhPCR.

[0008] The use of RNase H2 mutants, created through partial recombination of the amino acid sequence of wild-type PaRNase H2 with sequences from other related molecular species, has previously been shown to result in improved RNase H2 enzymatic activity in high-fidelity DNA polymerase buffers. Two of these mutants, Q48R SEL29 RNase H2 and Al07V SEL29 RNase H2, were shown to have improved enzymatic activity when using Thermococcus kodakarensis (KOD) DNA polymerase reaction buffer. In addition, both mutants show enhanced discrimination of mismatches opposite the RNA base as well as at the 3' and 5' ends of the RNA base compared to wild-type PaRNase H2. See U.S. Provisional Patent Application No. 63 / 130,548, filed December 24, 2020, and entitled "RNASE H2 MUTANTS THAT ENHANCE MISMATCH DISCRIMINATION AND ACTIVITY IN HIGH-FIDELITY POLYMERASE BUFFER" (Attorney Docket No. IDT01-018-PRO), the contents of which are incorporated by reference in their entirety into this specification. [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. Provisional Patent Application No. 63 / 130548 [Non-patent literature]

[0010] [Non-Patent Document 1] Eder, et al. (1993), Biochimie, 75, 123-126 Summary of the Invention [Problem to be solved by the invention]

[0011] This disclosure relates to one of these novel hybrid RNase H2 enzyme variants, Q48R SEL29 RNase H2, in a multiplex rhAmpSeq workflow containing high fidelity DNA polymerase and buffer. Compared to wild-type PaRNase H2, Q48R SEL29 RNase H2 reduces primer dimers generated during PCR amplification, thereby improving mapping rate and on-target rate. While improving these metrics, Q48R SEL29 RNase H2 has no effect on other critical sequencing metrics, including amplification product uniformity, amplification product dropout rate, and amplification product uniformity distribution. [Means for solving the problem]

[0012] Summary of the Invention In a first aspect, a hybrid RNase H2 protein is provided, the hybrid RNase H2 protein comprising fragments of amino acid sequences derived from the organisms Pyrococcus abyssi (Pa), Thermococcus kodakarensis (T. kod) and Pyrococcus furiosus.

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

[0014] In a third aspect, a method for performing primer extension is provided, the method comprising contacting a hybrid RNase H2 protein, as disclosed herein, with a primer, a polynucleotide template, nucleoside triphosphates and a DNA polymerase under conditions suitable for primer extension, thereby generating an extended primer.

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

[0016] In a fifth aspect, a method for performing rhPCR is provided, the method comprising the steps of performing primer extension with a hybrid RNase H2, a DNA polymerase and a primer as described herein.

[0017] In a sixth aspect, a method for amplifying a target DNA sequence is provided. The method includes several steps. The first step is to provide a reaction mixture including: (i) an oligonucleotide primer having a cleavage domain cleavable by an RNase H2 enzyme located 5' to a blocking group linked at or near the 3' end of the oligonucleotide primer, the blocking group preventing primer extension and / or preventing the oligonucleotide primer from being used 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 as 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 cleave the hybridized oligonucleotide primer with the hybrid RNase H2 enzyme at a cleavage site within or adjacent to the cleavage domain to remove the blocking group from the oligonucleotide primer.

[0018] In a seventh embodiment, a kit for generating an extension primer is provided. The kit comprises at least one container providing a hybrid RNase H2 protein as disclosed herein.

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

[0020] In a ninth aspect, a method for preparing an amplification product library of a template nucleic acid is provided. The method includes several steps. The first step is to form a mixture comprising a nucleic acid population, at least one blocked cleavable primer, a hybrid RNase H2 protein, dNTPs, a DNA polymerase, and a buffer, such that a hybrid duplex forms between the at least one blocked cleavable primer and the nucleic acid population in the mixture. The second step is to cleave the at least one blocked cleavable primer with a hybrid RNase H2 protein to generate at least one active primer capable of primer extension by a DNA polymerase. The third step is to extend the at least one active primer with a DNA polymerase in a buffer under conditions that allow amplification of one or more template nucleic acids from the nucleic acid population, thereby generating an amplification product 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 a second aspect, the DNA polymerase is KOD DNA polymerase or other high-fidelity archaeal DNA polymerase. In a third aspect, the buffer is a high-fidelity archaeal DNA polymerase buffer.

[0021] In a tenth aspect, a method for performing massively parallel sequencing is provided. The method includes several steps. The first step is to prepare a library population of template nucleic acids using a population of nucleic acids, a hybrid RNase H2 mutant protein, at least one blocked cleavable primer, a DNA polymerase, dNTPs and a buffer in a PCR method. The second step is to sequence a plurality of desired template nucleic acids from the library population of 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.

[0022] In an eleventh aspect, a method for detecting SNP-containing nucleic acid templates from an amplification product library of nucleic acid templates is provided. The method includes several steps. The first step includes forming a mixture including an amplification product library of nucleic acid templates; at least one blocked cleavable primer; a hybrid mutant RNase H2 protein; dNTPs; a DNA polymerase; and a buffer. A hybrid duplex is formed between the at least one blocked cleavable primer in the mixture and the SNP-containing nucleic acid template in the amplification product library of nucleic acid templates. The second step includes cleaving the at least one blocked cleavable primer of the hybrid duplex with a hybrid RNase H2 protein to generate at least one active primer capable of primer extension of the hybrid duplex by a DNA polymerase in a buffer solution under conditions that allow amplification of one or more template nucleic acids from the amplification product library of nucleic acid templates, thereby detecting the SNP-containing nucleic acid template. In a first aspect, the method includes a hybrid mutant RNase H2 protein selected from Q48R SEL29 (SEQ ID NO: 18) or other hybrid mutant RNase H2 protein. In a second aspect, the method includes a buffer that is a high fidelity archaeal DNA polymerase buffer.

[0023] In a twelfth aspect, a method for performing a loop-mediated amplification reaction is provided. The method includes two steps. The first step includes forming a mixture including a nucleic acid template; four blocked cleavable primers that form a duplex with the nucleic acid template that is a substrate for the RNase H2 protein; an RNase H2 protein selected from Q48R SEL29 (SEQ ID NO: 18) or other RNase H2 protein; a DNA polymerase protein; dNTPs; and a buffer. The second step includes performing an isothermal amplification cycle with the mixture.

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

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

[0026] [Figure 1A] FIG. 1 depicts an exemplary plot showing that dimer rate was reduced when using the mutant, Q48R SEL29 RNase H2 enzyme compared to the wild-type PaRNase H2 enzyme at all enzyme concentrations. [Figure 1B] FIG. 1 depicts exemplary data showing that mutant, Q48R SEL29 RNase H2 enzyme provides higher mapping rates compared to wild-type PaRNase H2 enzyme at all RNase H2 enzyme concentrations. [Figure 1C] FIG. 1 depicts exemplary data showing that the on-target rate with the mutant, Q48R SEL29 RNase H2 enzyme is higher compared to the wild-type PaRNase H2 enzyme at all enzyme concentrations. [Diagram 2] FIG. 1 depicts an example of the average normalized dimer counts per each primer pair identified, in which the mutant, Q48R SEL29 RNase H2 enzyme reduces the most primer dimers identified by half compared to the wild-type PaRNase H2 enzyme. [Figure 3A] FIG. 1 depicts exemplary data for amplicon uniformity of ≧0.2-fold and ≦0.05-fold (dropout rate), with library yields being comparable between mutant, Q48R SEL29 RNase H2 enzyme and wild-type PaRNase H2 enzyme at all concentrations. [Figure 3B] FIG. 1 depicts overall amplification product uniformity of ≧0.2 fold for the mutant, Q48R SEL29 RNase H2 enzyme is comparable to wild-type PaRNase H2 enzyme at various concentrations tested. [Figure 3C] FIG. 1 depicts that amplification product dropout rates are similar between the mutant, Q48R SEL29 RNase H2 enzyme and the wild-type PaRNase H2 enzyme for all titers tested. [Figure 4] FIG. 1 depicts exemplary data showing that the uniformity distribution (percentage of amplification products having coverage within the ranges of 0-0.1x, 0.1-0.2x, 0.2-0.5x, 0.5-1.5x, 1.5-2.5x, and 2.5-5x compared to the average coverage of the amplification products) is comparable between mutant, Q48R SEL29 RNase H2 enzyme and wild-type PaRNase H2 enzyme. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention provides novel hybrid RNase H2 enzyme variants that enhance enzymatic activity during rhPCR using certain DNA polymerase buffers while retaining their ability to retain or enhance mismatch discrimination in double-stranded templates. The RNase H2 enzyme hybrids combine fragments of amino acid sequences from the organisms Pyrococcus abyssi (Pa), Thermococcus kodakarensis (T. kod) and Pyrococcus furiosus. The resulting hybrid RNase H2 enzymes, as well as select mutants based on these enzymes, exhibit significantly enhanced mismatch discrimination. In particular, Q48R SEL29 RNase H2 (SEQ ID NO: 18) is shown to result in mixtures having a reduced population of primer dimer species compared to mixtures produced by wild-type PaRNase H2 (SEQ ID NO: 1) in, inter alia, methods of performing primer extension, performing rhPCR, amplifying target DNA sequences, performing massively parallel sequencing, detecting SNP-containing nucleic acid templates from an amplification product library of nucleic acid templates, and performing loop-mediated amplification reactions.

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

[0029] As used in the context of describing the present invention (especially in the context of the claims below), the terms "a," "an," "the," and similar referents shall be understood to cover both the singular and the plural, unless otherwise indicated herein or dictated to the contrary by context. The terms "comprising," "having," "including," and "containing" shall be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise noted. The recitation of ranges of values ​​herein is intended to be used only as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into this specification as if it were individually recited herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or dictated to the contrary by context. The use of any and all examples or exemplary phrases (e.g., "etc.") presented herein is intended only to better illustrate the invention and does not pose a limitation on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0030] As used herein, the terms "nucleic acid" and "oligonucleotide" refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose) and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base. No distinction in 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. Thus, these terms include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. For use in the present invention, oligonucleotides can also include nucleotide analogs modified at the base, sugar, or phosphate backbone, as well as non-purine or non-pyrimidine nucleotide analogs.

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

[0032] As used herein, the term "primer" refers to an oligonucleotide that can act as an initiation point for DNA synthesis under appropriate conditions. Such conditions include conditions in which synthesis of primer extension products that are complementary to a nucleic acid strand is induced in an appropriate buffer and at an appropriate temperature, in the presence of four different nucleoside triphosphates and an agent for extension (e.g., DNA polymerase or reverse transcriptase). Primer extension may also be performed in the absence of one or more of the nucleotide triphosphates, in which case an extension 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 "extended" by ligation with a second oligonucleotide that hybridizes at an adjacent position. Thus, as used herein, the term "primer extension" refers to both the polymerization of individual nucleoside triphosphates using a primer as an initiation point for DNA synthesis and the ligation of two oligonucleotides to form an extension product.

[0033] The primer is preferably a single-stranded DNA. The appropriate length of the primer depends on the intended use of the primer, but typically ranges from 6 to 50 nucleotides, preferably 15 to 35 nucleotides. Short 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 must be sufficiently complementary to the template to hybridize. The design of primers suitable for amplification is well known in the art and is described in the literature cited herein.

[0034] Primers may incorporate additional features that allow the primer to be detected or immobilized, but do not change the basic property of the primer, which is to act as a starting point for DNA synthesis. For example, primers may contain additional nucleic acid sequences at the 5' end that do not hybridize with the target nucleic acid, but that facilitate cloning or detection of the amplification product. In this specification, the region of the primer that is sufficiently complementary to the template to hybridize 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 a nucleic acid that is to be amplified, sequenced, or detected.

[0036] As used herein, the term "hybridization" refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between completely complementary nucleic acid strands, or between "substantially complementary" nucleic acid strands that contain small mismatched regions. Conditions under which the hybridization of completely complementary nucleic acid strands is strongly favored are called "stringent hybridization conditions" or "sequence-specific hybridization conditions". Stable duplexes with substantially complementary sequences can be achieved under less stringent hybridization conditions; however, the degree of mismatch allowed is controlled by appropriate adjustment of hybridization conditions. Those skilled in the art of nucleic acid technology may determine duplex stability empirically, following guidance provided by the art (see, e.g., 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), taking into account a number of variables, including, for example, the length and base pair composition of the oligonucleotide, ionic strength, and the occurrence of mismatched base pairs.

[0037] The term "amplification reaction" refers to any chemical reaction, including enzymatic reactions, that results in an increase in copies of a template nucleic acid sequence or results in transcription of a template nucleic acid. Amplification reactions include reverse transcription, polymerase chain reaction (PCR), including real-time PCR (see U.S. Pat. Nos. 4,683,195 and 4,683,202; see PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., 1990)), and ligase chain reaction (LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary "amplification reaction conditions" or "amplification conditions" typically include two-step or three-step cycles. Two-step cycles have a high temperature denaturation step followed by a hybridization / extension (or ligation) step. Three-step cycles include a denaturation step followed by a hybridization step followed by a separate extension or ligation 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 furiosus (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), 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 / Pyrococcus kodakaraensis KOD 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 may be determined by means well known in the art.

[0039] As used herein, a primer is "specific" for a target sequence if it hybridizes primarily to the target nucleic acid when used in an amplification reaction under sufficiently stringent conditions. Typically, a primer is specific for 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, such as salt conditions, primer base composition and mismatch position, affect the specificity of a primer, and confirmation of primer specificity through routine experimentation is often required. Hybridization conditions can be selected under which a primer can form a stable duplex only with a target sequence. Thus, the use of target-specific primers under appropriately stringent amplification conditions allows selective amplification of target sequences that contain the primer binding site of the target.

[0040] As used herein, the term "non-specific amplification" refers to the amplification of nucleic acid sequences other than the target sequence, resulting from primers hybridizing to sequences other than the target sequence and then being used as substrates for primer extension. Hybridization of primers to non-target sequences is called "non-specific hybridization" and tends to occur, inter alia, at low temperatures, low stringency, in pre-amplification conditions, or in the presence of mutant alleles in the sample that have sequences closely related to the true target, as in the case of single nucleotide polymorphisms (SNPs).

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

[0042] The term "3' mismatch discrimination assay" refers to an assay that identifies the presence of improved specificity of amplification of a target DNA sequence when the target DNA sequence is probed by two primers that have substantially identical sequences except for the occurrence of one or more nucleotide residues that have different base compositions at or near their respective 3' ends. For example, a first primer that has a 3' end sequence that is completely complementary to the target DNA sequence is considered to be a 3' match primer, and a second primer that has a 3' end sequence that has at least one nucleotide base that is non-complementary to the target DNA sequence is considered to be a 3' mismatch primer. Examples of 3' mismatch discrimination assays are provided in many of the examples, including, inter alia, Tables 10 and 11 of Example 4.

[0043] As used herein, the term "primer dimer" refers to a template-independent non-specific amplification product that may result from primer extension, where 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 also occur. As used herein, the term "primer dimer" is generally used to encompass template-independent non-specific amplification products.

[0044] As used herein, the term "reaction mixture" refers to a solution containing the reagents necessary to carry out a given reaction. An "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. A "PCR reaction mixture" typically contains oligonucleotide primers, DNA polymerase (most typically a thermostable DNA polymerase), dNTPs and divalent metal cations in a suitable buffer. A reaction mixture is said to be complete when it contains all the reagents necessary to enable a reaction, and incomplete when it contains only a subset of the necessary reagents. It will be understood by those skilled in the art that reaction components are by definition 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 reaction components are combined prior to the reaction to create a complete reaction mixture. Moreover, it will be understood by those of skill in the art that the reaction components may be packaged separately and that a useful commercial kit may contain any subset of the reaction components, including the protected primers of the present invention.

[0045] For the purposes of the present invention, the term "non-activated" or "inactivated" as used herein refers 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 a primer, the non-activated state occurs because the primer is protected at or near the 3' end to prevent primer extension. If a specific group is attached at or near the 3' end of the primer, DNA polymerase cannot bind to the primer and extension cannot occur. However, the non-activated primer is capable of hybridizing to a substantially complementary nucleotide sequence.

[0046] For the purposes of the present invention, the term "activated" as used herein refers to a primer or other oligonucleotide that is capable of participating in a reaction with DNA polymerase or DNA ligase. A primer or other oligonucleotide is activated after hybridizing to a substantially complementary nucleic acid sequence and is cleaved to provide a functional 3' or 5' end capable of interacting with DNA polymerase or DNA ligase. For example, if the oligonucleotide is a primer and the primer is hybridized to a template, the 3' blocking group is removed from the primer, for example, by a cleaving enzyme, so that DNA polymerase binds to the 3' end of the primer and promotes primer extension.

[0047] As used herein, the terms "cleavage domain" or "cleavable domain" are synonymous and refer to a region located between the 5' and 3' ends of a primer or other oligonucleotide that is recognized by a cleavage compound, e.g., a cleavage enzyme, that cleaves the primer or other oligonucleotide. For purposes of the present invention, the cleavage domain is designed to cleave the primer or other oligonucleotide only when it is hybridized to a complementary nucleic acid sequence and not when it is single-stranded. The cleavage domain or sequences adjacent thereto may contain a) a moiety that blocks or inhibits extension or ligation of the primer or other oligonucleotide by a polymerase or ligase, b) a moiety that enhances discrimination to detect mutant alleles, or c) a moiety that suppresses undesired cleavage reactions. One or more such moieties may be contained within the cleavage domain or within sequences adjacent thereto.

[0048] As used herein, the term "RNase H cleavage domain" is a type of cleavage domain that contains one or more ribonucleic acid residues or alternative analogs that provide a substrate for RNase H. The RNase H cleavage domain can be located anywhere within the primer or oligonucleotide, and is preferably located at or near the 3' end or at the 5' end of the molecule.

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

[0050] As used herein, the term "protected primer" refers to a primer that, at a minimum, possesses a cleavable domain suitable for sufficient hybridization with a target sequence, 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 term "cleavage compound" or "cleavage agent" refers to any compound that can recognize a cleavage domain in a primer or other oligonucleotide and selectively cleave an oligonucleotide based on the presence of the cleavage domain. The cleavage compounds utilized in the present invention selectively cleave primers or other oligonucleotides that contain a cleavage domain only when hybridized to 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 cleavage reaction preferred in the present invention results 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 is capable of recognizing a cleavage domain when a primer or other oligonucleotide is hybridized with a substantially complementary nucleic acid sequence, but does not cleave the complementary nucleic acid sequence (i.e., produces a single-stranded break within the duplex). A cleavage enzyme also does not cleave a primer or other oligonucleotide that contains a cleavage domain when 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 the double-stranded portion of a fully double-stranded nucleic acid or a partially double-stranded nucleic acid. The position where the 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 can be an enzyme or any other compound or composition. In certain embodiments, the nicking agent can 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 position (i.e., NS) relative to the position 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] Thus, as used herein, a "nicking endonuclease" (NE) refers to an endonuclease that recognizes a nucleotide sequence in a fully or partially double-stranded nucleic acid molecule and cleaves only one strand of the nucleic acid molecule at a specific location relative to the recognition sequence. In such cases, the entire sequence from the recognition site to the point of cleavage constitutes the "cleavage domain."

[0055] As used herein, the term "blocking group" refers to a chemical moiety attached to a primer or other oligonucleotide such that the amplification reaction does not occur. For example, primer extension and / or DNA ligation does not occur. When the blocking group is removed from the primer or other oligonucleotide, the oligonucleotide is able to participate in the assay for which it is designed (PCR, ligation, sequencing, etc.). Thus, a "blocking group" can be any chemical moiety that inhibits recognition by a polymerase or DNA ligase. A blocking group can generally be incorporated into a cleavage domain located 5' or 3' of the cleavage domain. A blocking group can be composed of more than one chemical moiety. In the present invention, a "blocking group" is typically removed after hybridization of the oligonucleotide with its target sequence.

[0056] The term "fluorogenic probe" refers to a) an oligonucleotide conjugated with a fluorophore and a quencher, and, optionally, a minor groove binder or b) a DNA binding reagent such as SYBR® Green dye.

[0057] The term "fluorescent label" or "fluorophore" refers to a compound having a fluorescence emission maximum between about 350 and 900 nm. Examples of such compounds include 5-FAM (also called 5-carboxyfluorescein; also called Spiro(isobenzofuran-1(3H),9'-(9H)xanthene)-5-carboxylic acid,3',6'-dihydroxy-3-oxo-6-carboxyfluorescein); 5-hexachloro-fluorescein; ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloyl-fluoresceinyl)-6-carboxylic acid]); 6-hexachloro-fluorescein; 5-Tetrachloro-fluorescein;([4,7,2',4',5',7'-Hexachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]);5-Tetrachloro-fluorescein;([4,7,2',7'-Tetrachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]);6-Tetrachloro-fluorescein;([4,7,2',7'-Tetrachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid] Xanthylium; 6-TAMRA(6-carboxytetramethylrhodamine); 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid) A wide variety of fluorophores may be used, including, but not limited to, 1,5-IAEDANS (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid); Cy5 (indodicarbocyanine-5); Cy3 (indo-dicarbocyanine-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 dyes; Max dyes (Integrated DNA Technologies), as well as suitable derivatives thereof.

[0058] As used herein, the term "quencher" refers to a molecule or moiety of a compound that is capable of reducing emission from a fluorescent donor when bound to or in close proximity to the donor. Quenching can occur by any of several mechanisms, including fluorescence resonance energy transfer, photoinduced electron transfer, paramagnetic intersystem crossing enhancement, excitation coupling, such as Dexter exchange coupling, and formation of dark complexes. Fluorescence is "quenched" when the fluorescence emitted by the fluorophore is reduced by at least 10%, e.g., 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9% or more, compared to the fluorescence in the absence of the quencher. A number of commercially available quenchers are known in the art, 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 have no natural fluorescence and virtually 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 joining of two polynucleotide ends. In various embodiments, ligation involves the covalent joining of the 3' end of a first polynucleotide (acceptor) to the 5' end of a second polynucleotide (donor). Ligation results in a phosphodiester bond forming between the polynucleotide ends. In various embodiments, ligation can be mediated by any enzyme, chemical reaction, or process that results in the covalent joining of 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. coli DNA ligase, T4 DNA ligase, and the like.

[0061] Ligation reactions can also be used in DNA amplification methods such as "ligase chain reaction" (LCR), also called "ligase amplification reaction" (LAR) (see also Barany, Proc. Natl. Acad. Sci., 88:189 (1991); and Wu and Wallace, Genomics, 4:560 (1989), incorporated herein by reference). In LCR, four oligonucleotides are mixed, two adjacent oligonucleotides that hybridize uniquely to one strand of target DNA and a complementary set of adjacent oligonucleotides that hybridize to the opposite strand, and DNA ligase is added to the mixture. In the presence of the target sequence, DNA ligase covalently links each set of hybridized molecules. Importantly, in LCR, two oligonucleotides are ligated together only if they base-pair with the sequence without gaps. Repeated cycles of denaturation, hybridization, and ligation amplify short segments of DNA. Mismatches at the junctions between adjacent oligonucleotides inhibit ligation. In other oligonucleotide ligation assays, this property allows LCR to be used to distinguish mutant alleles such as SNPs. LCR has also been used in combination with PCR to achieve enhanced detection of single base changes (see Segev, PCT Publication No. WO9001069 (1990)).

[0062] The term "codon optimization" refers to the incorporation of codons that modify a particular nucleic acid encoding a polypeptide, such that it is preferred for efficient expression in a given host cell, such as a given microbial cell (e.g., E. coli, S. cerevisae, among others) or mammalian cell (e.g., human cells such as HeLa cells, COS cells, among others). Such preferred codons are well known in the art based on codon bias tables that have been developed for various organisms. Polynucleotides encoding the polypeptides of the 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 discerned from empirical determination.

[0063] The phrase "BaseX PCR amplification" refers to a highly efficient nucleic acid amplification method that allows for greater than two-fold increase in amplification product with each amplification cycle, thereby allowing for increased sensitivity and speed over conventional PCR. This method is disclosed in U.S. Patent Publication No. US10273534(B2), entitled "Exponential base-greater-than-2 nucleic acid amplification" by R. Higuchi (applicant: Cepheid), published April 30, 2019, the contents of which are incorporated herein by reference in their entirety.

[0064] The phrase "fusion protein" or "fusion polypeptide" refers to the incorporation of additional amino acid information, not native to the protein, covalently attached to the protein. Such additional amino acid information may include a tag that allows purification or identification of the fusion protein. Such additional amino acid information may include a peptide that allows the fusion protein to be transported into the cell and / or to a specific location within the cell. Examples of tags for these purposes are: AviTag, a peptide that allows biotinylation by the enzyme BirA so that the protein can be isolated with streptavidin; Calmodulin tag, a peptide that is bound by the protein calmodulin; Polyglutamic acid tag, a peptide that binds efficiently to anion exchange resins such as Mono-Q; E tag, a peptide that is recognized by an antibody; FLAG tag, a peptide that is recognized by an antibody; HA tag, a peptide derived from hemagglutinin that is recognized by an antibody; and TATA, a peptide that is typically a nickel chelator or a cobalt chelator. His tag, which is 5-10 histidines bound by a chelating agent; Myc tag, which is a peptide derived from c-myc recognized by an antibody; NE tag, which is a novel 18 amino acid synthetic peptide recognized by a monoclonal IgG1 antibody and useful in a wide range of applications including Western blotting, ELISA, flow cytometry, immunohistochemistry, immunoprecipitation and affinity purification of recombinant proteins; S tag, which is a peptide derived from ribonuclease A; SBP tag, which is a peptide that binds to streptavidin; Softag tag, which is intended for expression in mammals. 1; Softag, intended for expression in prokaryotes; Strep tag, a peptide that binds to a modified streptavidin called streptavidin or streptactin (Strep tag II); TC tag, a biarsenical tetracysteine ​​tag recognized by the FlAsH and ReAsH biarsenicals; V5 tag, a peptide recognized by an antibody; VSV tag, a peptide recognized by an antibody; Xpress tag; Isopeptag, a peptide that covalently binds to the pilin C protein;the SpyTag, a peptide that covalently binds to the SpyCatcher protein; the SnoopTag, a peptide that covalently binds to the SnoopCatcher protein; the BCCP (biotin carboxyl carrier protein), a protein domain biotinylated with BirA to allow recognition by streptavidin; the glutathione-S-transferase tag, a protein that binds to immobilized glutathione; the green fluorescent protein tag, a protein that is spontaneously fluorescent and can be bound by an antibody; the HaloTag, a mutant bacterial haloalkane dehalogenase that covalently conjugates to reactive haloalkane substrates, allowing conjugation to a wide variety of substrates; the maltose binding protein tag, a protein that binds to amylose agarose; the Nus tag; the thioredoxin tag; and the Fc tag, derived from an immunoglobulin Fc domain, which allows dimerization and solubilization and can be used for purification on Protein A Sepharose. A nuclear localization signal (NLS), such as the NLS derived from SV40, allows the protein to be transported to the nucleus immediately after entering the cell. Given that the native Cas9 protein is of bacterial origin and therefore does not naturally contain an NLS motif, the addition of one or more NLS motifs to a recombinant Cas9 protein is expected to show improved genome editing activity when used in eukaryotic cells where the DNA substrate of the target genome resides in the nucleus. Those skilled in the art will understand these various fusion tag technologies, the specific amino acid sequences involved, as well as how to make and use fusion proteins containing them. In one embodiment, a highly preferred fusion protein or fusion polypeptide contains a His tag motif, although those skilled in the art will understand that other tags may also be included, as mentioned above. The present invention includes fusion proteins or fusion polypeptides, as well as original mutant forms of the corresponding proteins or polypeptides that lack additional amino acid sequence information;

[0065] Novel hybrid RNase H2 enzyme variants generated 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 from three RNase H2 enzymes, including Pyrococcus abyssi, Thermococcus kodakarensis, and Pyrococcus furiosus, 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), combining fragments of amino acid sequences from the organisms Pyrococcus abyssi, Thermococcus kodakarensis, and Pyrococcus furiosus, were found to exhibit significantly enhanced mismatch discrimination. The mutants were selected from a library created by random shuffling of RNase H2 sequences. Although other changes are also present, both mutant enzymes contain a fragment of amino acid residues 26-40 and residues 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 contact the bound DNA duplex. Without being bound to any particular theory, it can be hypothesized that residues 26-40 and residues 100-120 of T. kod RNase H2 alter the enzyme binding pocket for the substrate duplex, resulting in altered binding affinity and catalysis of nucleic acid cleavage. The amino acid sequences of the wild-type P.ab. RNase H2 protein (SEQ ID NO: 88), the hybrid SEL28 RNase H2 protein (SEQ ID NO: 89), and the hybrid SEL29 RNase H2 protein (SEQ ID NO: 90) are depicted in Table 1. The corresponding (His)6-tagged amino acid sequences of 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 basis for generating additional mutant RNase H2 proteins (see Table 3).

[0066] [Table 1] TIFF2024501832000003.tif133166

[0067] The resulting hybrid SEL28 and SEL29 RNase H2 enzymes encoded by SEQ ID NOs:89 and 90 improve mismatch discrimination when the mismatch is placed at an RNA nucleotide, but to varying degrees and with different specificities (data not shown). Similarly, the SEL28 and SEL29 mutant RNase H2 enzymes encoded by SEQ ID NOs:89 and 90 improve mismatch discrimination at the 5' side of an RNA nucleotide (data not shown).

[0068] rhPCR could also be performed using high-fidelity DNA polymerases (such as DNA polymerases from Pyrococcus furiosus and Thermococcus kodakarensis (KOD)) rather than DNA polymerase from Thermus aquaticus (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 can be found that have a greater tolerance to components in the reaction buffer for KOD DNA polymerase. We show that Q48R, A107V and P13S / A107V, when added to the hybrid mutants, SEL28 RNase H2 or SEL29 RNase H2, enhance enzyme activity using KOD DNA polymerase reaction buffer.

[0069] The present invention relates to mutant RNase H2 enzymes that have enhanced enzymatic 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. A screen of seven point mutants with a SEL28 RNase H2 or SEL29 RNase H2 background was performed and showed that Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 have enhanced enzymatic activity using KOD DNA polymerase reaction buffer. They were also shown to retain or enhance the mismatch discrimination 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 to increase PCR specificity and eliminate primer dimers by using RNase H2 from Pyrococcus abyssi or related organisms and a DNA primer containing a single ribonucleotide residue and a 3'-protecting portion ("blocked cleavable primer"). The blocked cleavable primer is activated when cleaved by the RNase H2 enzyme. Cleavage occurs at the 5' side of the RNA base after hybridization of the primer with the target DNA. Primer dimers are reduced because the primer can be cleaved only after hybridization with a perfectly matched target sequence. The requirement for high target complementarity reduces the amplification of closely related sequences.

[0071] In this regard, the hybrid RNase H2 mutant proteins are particularly suitable for enhanced performance in generating high quality genomic amplicon libraries for high throughput multiplexed sequencing applications, such as next generation sequencing applications (NGS). In particular, Q48R SEL29 is an RNase H2 enzyme useful for RNase H2-mediated PCR applications and systems such as Applicants' rhAmpSeq™ System. RNase H2-mediated SNP detection and RNase H2-mediated rare allele detection Due to the enhanced mismatch discrimination attributes at the 3' side, the hybrid RNase H2 mutant proteins disclosed herein can be used for single nucleotide polymorphism detection and rare allele detection. The use of blocked cleavable primers that form a perfect duplex only with a 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 duplex for primer extension by a DNA polymerase under appropriate conditions.

[0072] RNase H2 in loop-mediated isothermal amplification (LAMP) The use of RNase H2 in loop-mediated isothermal amplification (LAMP) is also envisaged herein. LAMP amplification 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 amplicon (Notomi et al., Nucleic Acids Research, 28(12)(2000)). The amplification reaction relies on the strand displacement activity of DNA polymerase, usually from Bacillus stearothermophilus (Bst). The product has a structure consisting of a long inverted repeat of the target sequence.

[0073] LAMP reactions are prone to the formation of primer dimer products due to the use of many primers and mesophilic DNA polymerases in the method. LAMP also lacks 5' to 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 LAMP reactions. In this regard, hybrid RNase H2 mutant proteins are particularly suitable for enhancing the performance of the desired products formed in LAMP reactions without the concomitant generation of primer dimers.

[0074] Applicable In a first aspect, there is provided a hybrid RNase H2 protein. The hybrid RNase H2 protein comprises a fragment of an amino acid sequence from the organisms Pyrococcus abyssi (Pa), Thermococcus kodakarensis (T. kod) and Pyrococcus furiosus. In a first related aspect, the hybrid RNase H2 protein comprises amino acid residues 26-40 and residues 100-120 of T. kod RNase H2. In a second related aspect, the hybrid RNase H2 protein is selected from SEQ ID NOs: 2 and 3. In a third related aspect, the hybrid RNase H2 protein is selected from SEQ ID NOs: 14-20.

[0075] In a second aspect, there is provided a recombinant nucleic acid encoding any of the hybrid RNase H2 proteins disclosed herein. In a first related aspect, an exemplary recombinant nucleic acid encoding any of the hybrid RNase H2 proteins includes SEQ ID NOs: 79-87 of Table 14.

[0076] In a third aspect, a method for performing primer extension is provided. The method comprises contacting a hybrid RNase H2 protein disclosed herein with a primer, a polynucleotide template, a nucleoside triphosphate and a DNA polymerase under conditions suitable for the primer extension method, thereby generating an extended primer. In a first aspect, the DNA polymerase comprises a high-fidelity archaeal DNA polymerase. In a second aspect, the primer comprises a blocked cleavable primer. In a third aspect, the primer extension method comprises a method for performing a polymerase chain reaction (PCR). In a fourth aspect, the method for performing PCR improves mismatch discrimination in a primer:polynucleotide hybrid formed between a primer and a polynucleotide template. In a fifth aspect, the improved mismatch discrimination comprises improved mismatch discrimination at the 3' side.

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

[0078] In a fifth aspect, a method for performing rhPCR is provided. The method comprises performing primer extension with a hybrid RNase H2, a DNA polymerase and a primer as described herein. In a first aspect, the method for performing rhPCR comprises performing primer extension with a high-fidelity archaeal DNA polymerase. In a second aspect, the hybrid RNase H2 enzyme is reversibly inactivated by chemical modification, an aptamer or a blocking antibody. In a third aspect, a blocking group is attached to the 3'-terminal nucleotide of the primer. In a fourth aspect, a blocking group is attached to the 5'-side of the 3'-terminal residue to prevent the primer from being used as a template for DNA synthesis. In a fifth aspect, the blocking group comprises one or more abasic residues. In a sixth aspect, the one or more abasic residues are C3 spacers. In a seventh related aspect, the blocking group comprises one member selected from the group consisting of RDDDDx, RDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD, where R is an RNA residue, D is a DNA residue, M is a mismatch residue, and x is a C3 spacer or other proprietary group that is resistant to degradation. In this respect, the blocking group comprises a label that allows detection of the extension amplification reaction. In this respect, a label that allows detection of the amplification reaction is attached to the oligonucleotide primer 3' from the cleavage site. In this respect, the label is a fluorophore or a mass tag for detection of the amplification reaction by mass spectrometry. In a further embodiment, the cleavage domain of the blocked cleavable primer comprises one or more of the following moieties: a DNA residue, an abasic residue, a modified nucleoside, or an internucleotide modified phosphate linkage. In further related aspects, the cleavage domain can comprise a single RNA residue, two adjacent RNA residues, a contiguous sequence of three or more RNA residues, and lacks an RNA residue or one or more 2' modified nucleosides.In related matters where the cleavage domain comprises one or more 2' modified nucleosides, the one or more 2' modified nucleosides are selected from the group consisting of 2'-O-alkyl RNA nucleosides, 2'-fluoro nucleosides, locked nucleic acids, 2'-ethylene nucleic acid residues, 2'-alkyl nucleosides, 2'-amino nucleosides and 2'-thionucleosides. Exemplary 2' modified nucleosides include 2'-O-methyl RNA nucleosides and 2'-fluoro nucleosides.

[0079] In a sixth aspect, a method for amplifying a target DNA sequence is provided. The method includes several steps. The first step is to provide a reaction mixture including: (i) an oligonucleotide primer having a cleavage domain cleavable by an RNase H2 enzyme located 5' to a blocking group linked at or near the 3' end of the oligonucleotide primer, the blocking group preventing primer extension and / or preventing the oligonucleotide primer from being used 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 as 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 cleave the hybridized oligonucleotide primer with the hybrid RNase H2 enzyme at a cleavage site within or adjacent to the cleavage domain to remove the blocking group from the oligonucleotide primer. In a first related subject matter of the method, the DNA polymerase is an archaeal high fidelity DNA polymerase. In a second related subject matter, the RNase H2 protein is reversibly inactivated by chemical modification or a blocking antibody. In a further related subject matter of the method, a blocking group is attached to the 3' terminal nucleotide of the oligonucleotide primer. In a further related subject matter of the method, a blocking group is attached to the 5' side of the 3' terminal residue to prevent the oligonucleotide primer from being used as a template for DNA synthesis. In a further related subject matter of the method, the blocking group comprises one or more abasic residues. In a further related subject matter of the method, the one or more abasic residues are a C3 spacer or other proprietary group that is resistant to degradation.In further aspects of the method, the blocking group comprises a member selected from the group consisting of RDDDDx, RDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD, where R is an RNA residue, D is a DNA residue, M is a mismatch residue and x is a C3 spacer or other proprietary group that is resistant to degradation. In further aspects of the method, the blocking group comprises a label that allows for detection of the extension amplification reaction. In further aspects of the method, the method further comprises a label that allows for detection of the amplification reaction, where the label is attached to the oligonucleotide primer 3' from the cleavage site. In these aspects, the label is a fluorophore or a mass tag for detection of the amplification reaction by mass spectrometry. In further aspects of the method, the cleavage domain comprises one or more of the following moieties: a DNA residue, an abasic residue, a modified nucleoside or an internucleotide modified phosphate linkage. In further aspects of the method, the cleavage domain comprises a single RNA residue, two adjacent RNA residues, a consecutive sequence of three or more RNA residues, or one or more 2'-modified nucleosides. In aspects where the cleavage domain comprises one or more 2'-modified nucleosides, the 2'-modified nucleosides are selected from the group consisting of 2'-O-alkyl RNA nucleosides, 2'-fluoro nucleosides, locked nucleic acids, 2'-ethylene nucleic acid residues, 2'-alkyl nucleosides, 2'-amino nucleosides, and 2'-thio nucleosides. Exemplary 2'-modified nucleosides include 2'-O-methyl RNA nucleosides and 2'-fluoro nucleosides.

[0080] In a seventh aspect, a kit for making an extension primer is provided. The kit comprises at least one container that provides the hybrid RNase H2 protein disclosed herein. In a first aspect, the kit further comprises one or more additional containers selected from the group consisting of: (a) a container that provides a primer that is hybridizable with a predetermined polynucleotide template under extension conditions; (b) a container that provides nucleoside triphosphates; (c) a container that provides a buffer suitable for primer extension, and (d) a DNA polymerase. In a second aspect, the DNA polymerase comprises a high fidelity archaeal DNA polymerase. In a third aspect, the aforementioned kit comprises one or more additional containers that contain a blocked cleavable primer.

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

[0082] In a ninth aspect, a method for preparing an amplification product library of a template nucleic acid is provided. The method includes several steps. The first step is to form a mixture comprising a nucleic acid population, at least one blocked cleavable primer, a hybrid RNase H2 protein, dNTPs, a DNA polymerase, and a buffer, such that a hybrid duplex is formed between the at least one blocked cleavable primer and the nucleic acid population in the mixture. The second step is to cleave the at least one blocked cleavable primer with a hybrid RNase H2 protein to generate at least one active primer capable of primer extension by a DNA polymerase. The third step is to extend the at least one active primer with a DNA polymerase in a buffer under conditions that allow amplification of one or more template nucleic acids from the nucleic acid population, thereby generating an amplicon 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 a second aspect, the DNA polymerase is a high-fidelity archaeal DNA polymerase or other DNA polymerase. In a third aspect, the buffer is a high-fidelity archaeal DNA polymerase buffer.

[0083] In a tenth aspect, a method for performing massively parallel sequencing is provided. The method includes several steps. The first step is to prepare a library population of template nucleic acids using a population of nucleic acids, a hybrid RNase H2 mutant protein, at least one blocked cleavable primer, a DNA polymerase, dNTPs and a buffer in a PCR method. The second step is to sequence a plurality of desired template nucleic acids from the library population of 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.

[0084] In an eleventh aspect, a method for detecting SNP-containing nucleic acid templates from an amplification product library of nucleic acid templates is provided. The method includes several steps. The first step includes forming a mixture including an amplification product library of nucleic acid templates; at least one blocked cleavable primer; a hybrid mutant RNase H2 protein; dNTPs; a DNA polymerase; and a buffer. A hybrid duplex is formed between the at least one blocked cleavable primer in the mixture and the SNP-containing nucleic acid template in the amplification product library of nucleic acid templates. The second step includes cleaving the at least one blocked cleavable primer of the hybrid duplex with a hybrid RNase H2 protein to generate at least one active primer capable of primer extension of the hybrid duplex by a DNA polymerase in a buffer solution under conditions that allow amplification of one or more template nucleic acids from the amplification product library of nucleic acid templates, thereby detecting the SNP-containing nucleic acid template. In a first aspect, the method includes a hybrid mutant RNase H2 protein selected from Q48R SEL29 (SEQ ID NO: 18) or other hybrid RNase H2 proteins. In a second aspect, the method includes a buffer that is a high fidelity archaeal DNA polymerase buffer.

[0085] In a twelfth aspect, a method for performing a loop-mediated amplification reaction is provided. The method includes two steps. The first step includes forming a mixture including a nucleic acid template; four blocked cleavable primers that form a duplex with the nucleic acid template that 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 protein; a DNA polymerase protein; dNTPs; and a buffer. The second step includes performing an isothermal amplification cycle with the mixture.

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

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

[0088] Finally, the RNase H2 polypeptides of the present invention are suitable for use in the BaseX PCR amplification method, a highly efficient amplification method disclosed in U.S. Patent Publication No. US10273534(B2), the contents of which are incorporated herein by reference in their entirety. EXAMPLES

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

[0090] Example 1: Generation of SEL28 and SEL29 hybrid RNase H2 proteins via recombinant reshuffling Mutant RNase H2 proteins were synthesized using in vitro DNA recombination and directed molecular evolution. A library of 5,500 mutants was generated by Altravax™ Inc. (Sunnyvale, CA) under a contractual agreement with Integrated DNA Technologies. Initial screening at IDT selected SEL28 and SEL29 mutants, which exhibited increased mismatch discrimination in the RNase H2 cleavage reaction. Mutants were created in the pET-27b(+) plasmid vector within Escherichia coli (E. coli) BL21(DE3). The protein expressed based on the T7 system contains the 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. coli cells were grown in 12 mL LB broth with 50 μg / mL kanamycin (Teknova™, Hollister, CA) using 50 mL TPP TubeSpin® Bioreactors (Techno Plastic Products AG, Trasadingen, Switzerland). Expression of RNase H2 was induced using an overnight Express™ Autoinduction System 1 (MilliporeSigma™, Burlington, MA) at 37° C. for 20 hours. Bacterial expression strains were grown with shaking at 250 rpm using a MaxQ™ 4000 orbital shaker (ThermoFisher Scientific™, Grand Island, NY). Cells were centrifuged at 7,500×g in a ThermoScientific Sorvall™ Legend XTR centrifuge for 10 minutes and the supernatant was discarded.The cell paste was stored at -80°C and resuspended in 0.6 mL of lysis solution consisting 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). Lysis took 15 minutes at 25°C with cells shaking at 120 rpm. Insoluble material was removed by centrifugation at 16,000×g for 20 min. DNase I and native E. coli proteins were denatured at 75° C. for 15 min. Insoluble denatured proteins were again removed by centrifugation at 16,000×g for 15 min. For protein purification, the Capturem™ His-Tagged Miniprep Kit (Takara Bio™, Mountain View, CA) was used. The binding 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 min. The column was washed twice with 200 μL of wash buffer (20 mM Na3PO4, 150 mM NaCl, pH 7.6) supplemented with 20 mM imidazole.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 against 1 L of 2x 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 dialysis buffer in the dialysis tank was replaced at least once. Samples were collected from dialysis and mixed with a mixture of 99.8% (v / v) glycerol and 0.2% (v / v) TritonX-100 in a 1:1 volume ratio. These purified and concentrated RNase H2 solutions were stored at -20°C. Their purity was estimated by SDS gel electrophoresis using Any kD™ Mini-PROTEAN® TGX Stain-Free™ Protein Gels (Bio-Rad®, Hercules, CA). The RNase H2 enzyme represented the major protein band (>75%) in all purified samples, the position of which 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 NO: 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 was collected using an Applied Biosystems 3130 Genetic Analyzer.

[0092] Example 2: The Q48R and A107V SDM mutants in SEL29 RNase H2 increase enzyme activity in Thermococcus kodakarensis DNA polymerase reaction buffer compared to SEL29 RNase H2 (His)6-tagged mutants of SEL28 and SEL29 RNase H2 proteins were generated by site-directed mutagenesis (SDM) (see, e.g., Weiner M. et al., Gene, 151:119-123 (1994)). Primers used for SDM of SEL28 and SEL29 RNase H2 are shown in Table 2, SEQ ID NOs: 4-13. The sequences of the mutants were verified and the proteins were expressed in E. coli using standard methods. Purification was performed using charged Ni-peptide as previously described (Dobosy et al., 2011; and U.S. Patent No. 8,911,948 B2). 2+ 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] TIFF2024501832000006.tif252163TIFF2024501832000007.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 have increased activity in KOD DNA polymerase reaction buffer compared to WT PaRNase H2, a quantitative rhPCR assay was designed targeting the rs4939827 SNP in SMAD7. This SNP has been previously utilized to characterize the efficiency and specificity of rhPCR (Dobosy et al., 2011; and U.S. Patent No. 8,911,948 B2) 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 10 μL reaction volume. Thermal cycling and data collection were performed on a CFX384® Real Time System (Bio-Rad®, Hercules, Calif.). Briefly, 200 nM (2 pmoles) of protected forward primer (SEQ ID NO:22) and 200 nM (2 pmoles) of unblocked reverse primer (SEQ ID NO:23) or 200 nM (2 pmoles) of unblocked forward primer (SEQ ID NO:21) and 200 nM (2 pmoles) of unblocked reverse primer (SEQ ID NO:23) were mixed into 1x internal KOD buffer (ROKStar buffer v2.0 or v1.66) (IDT, Coralville, IA) with 2.5 mM (total concentration) MgSO4, 0.2 mM (each concentration) of dNTPs (MilliporeSigma™, Burlington, MA) and 0.5x concentration of EvaGreen® dye (Biotium Inc., Fremont, CA).RNase H2 Dilution Buffer (IDT, Coralville, IA) or 21 femtomoles 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. 10 ng of genomic DNA of a cell line (cell line, NA12878; Coriell Institute for Medical Research, Camden, NJ) representing a homozygous genotype at the rs4939827 SNP was added to each reaction. Reactions were performed in triplicate and results were averaged. Reactions were performed under the following conditions: 95°C. 3:00 →(95℃ 0:10 →60℃ 0:30 )×75. Fluorescence data for intercalated EvaGreen® was collected after each extension time point. After the assay was completed, the data was analyzed and the CFX Manager® software's automated decision making function was used to determine the CFX ratio. q The values ​​were calculated and the results are presented in Table 5.

[0096] [Table 4]

[0097] [Table 5] TIFF2024501832000010.tif180162

[0098] These data indicate that Q48R SEL29 RNase H2 and A107V SEL29 RNase H2, but not P13S SEL29 RNase H2 and P13S / A107V SEL29 RNase H2, increased enzyme activity in Thermococcus kodakarensis DNA polymerase reaction buffer compared to background SEL29 RNase H2. In addition, P13S SEL28 RNase H2, A107V SEL28 RNase H2, and P13S / A107V SEL28 RNase H2 do not increase enzyme activity in Thermococcus kodakarensis DNA polymerase reaction buffer compared to background SEL28 RNase H2. Unless RNase H2 is added, the blocked primers are not cleaved and therefore cannot support PCR. The use of proprietary exonuclease-resistant blocked primers is necessary to prevent deprotection of the primers by high-fidelity DNA polymerases. WT(Pa)RNase H2 is able to cleave blocked primers, resulting in delayed amplification with a ΔC of 12.4 cycles in ROKstar buffer v2.0 and 21.1 cycles in ROKstar buffer v1.66 ​​compared to unblocked primers. q The delay in amplification, ΔC q Quantification of the amplification delay 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. qQuantification of ΔC for amplification delay is reduced 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 reduces ΔC for amplification delay. q Quantification of amplification time increases to 26.9 cycles in ROKstar buffer v2.0 and 32.8 cycles in ROKstar buffer v1.66, whereas 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 exhibits a ΔC q Quantification of the amplification delay is increased to 21.5 cycles in ROKstar buffer v2.0 and 31.8 cycles in ROKstar buffer v1.66. A107V SEL28 RNase H2 increases the ΔC q quantification to 20.0 cycles in ROKstar buffer v2.0 and 35.8 cycles in ROKstar buffer v1.66. These results are in contrast to the mutations in PaRNase H2, as Q48R PaRNase H2, A107V PaRNase H2 and P13S / A107V PaRNase H2 all increased enzyme activity in Thermococcus kodakarensis DNA polymerase reaction buffer, and P13S PaRNase H2 also had low, but measurable, enzyme activity in Thermococcus kodakarensis DNA polymerase reaction buffer. Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 improve enzyme activity (albeit to different extents) when used with Thermococcus kodakarensis DNA polymerase reaction buffer.

[0099] Example 3: Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 increase mismatch discrimination compared to WT RNase H2 when mismatches are located on opposite sides of the RNA The specific activity of the enzyme was determined using a fluorescence-based kinetic assay. The sequences for the DNA substrates are shown in Table 6, SEQ ID NOs: 24-25. The substrates are DNA hairpins with matched RNA bases within the double-stranded region. At the 3' end of the probe, 6-FAM (6-carboxyfluorescein) is conjugated; at the 5' end of the probe, Iowa Black® FQ (SEQ ID NO: 24) is conjugated. In intact hairpin probes, the fluorescence of 6-FAM is quenched by the Iowa Black® FQ moiety. RNase H2 cleaves and releases the 3' end of the probe with 6-FAM at the 5' side of the RNA base. Thus, the fluorescence of 6-FAM is no longer quenched and can fluoresce. A DNA hairpin with an RNA base but no fluorophore or quencher was used as a competitor (SEQ ID NO: 25). The assay was performed in a 10 μL reaction volume. Data collection was performed on a LightCycler® 480 II (Roche Life Science, Indianapolis, IN). Briefly, rhAmp™ Backbone 1× at 1× concentration was used to detect 6-FAM fluorescence. v3 was used in combination with 200 nM (2 pmoles) of labeled hairpin (SEQ ID NO:24) and 10 μM (100 pmoles) of competitor 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 NO:18-19) were added to each reaction. Reactions were initially kept at 4°C to prevent substrate cleavage prior to initiating the assay. Samples were run at 65°C. Fluorescence excitation wavelength was 483 nm; fluorescence emission wavelength was 533 nm. Fluorescence intensity was collected every 13.75 seconds over a period of 135 minutes. For each reaction, the initial rate and rate per femtomole were calculated. For each mutant, the rate per femtomole was calculated based on the WT, previously determined to have a specific activity of 17 units per μg of enzyme. Normalized against values ​​for PaRNase H2.Q48R SEL29 RNase H2 has a specific activity of 46.03 units / μg of enzyme and A107V SEL29 RNase H2 has a specific unit activity of 7.77 units / μg of enzyme.

[0100] [Table 6]

[0101] To fully determine whether these mutant RNase H2 enzymes improve mismatch discrimination for mismatches directly opposite an RNA base, a synthetic, quantitative rhPCR assay was utilized as previously described (Dobosy et al., 2011; and U.S. Patent No. 8,911,948 B2). This assay allows the effect of each specific, single-base mismatch to be directly compared to 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 10 μL reaction volume. Thermal cycling and data collection were performed on a CFX384® Real Time System (Bio-Rad®, Hercules, CA). Briefly, 200 nM (2 pmoles) of blocked reverse primer (SEQ ID NO:28-31) and 200 nM (2 pmoles) of unblocked forward primer (SEQ ID NO:26) or 200 nM (2 pmoles) of unblocked reverse primer (SEQ ID NO:27) and 200 nM (2 pmoles) of unblocked forward primer (SEQ ID NO:26) were mixed into a 1× concentration of iQ™ SYBR® Green Supermix® (Bio-Rad®, Hercules, Calif.). 5 mU of WT Pa (SEQ ID NO:1) RNase H2 enzyme, 40 mU of Q48R SEL29 (SEQ ID NO:18) RNase H2 enzyme or 5 mU of A107V SEL29 (SEQ ID NO:19) RNase H2 enzyme were added to each reaction. 20,000 copies of each synthetic target sequence (SEQ ID NOs: 32-35), each with a different nucleotide directly opposite the RNA base, were added to each reaction. Reactions were performed in triplicate and the results were averaged. Reactions were run under the following conditions: 95°C, 100°F, 50°C, 100°F, 100°C, 200°C, 50°C, 100°C, 200°C, 200°C, 5 ... 3:00 →(95℃ 0:10 →60℃ 0:30 )×75. Fluorescence data for the intercalated SYBR® Green was collected after each extension time point. After the assay was completed, the data was analyzed and the average C q value and ΔC qThe values ​​were calculated and the results are presented in Tables 8 and 9.

[0102] [Table 7]

[0103] [Table 8] TIFF2024501832000014.tif140168TIFF2024501832000015.tif139167

[0104] [Table 9] TIFF2024501832000017.tif118168TIFF2024501832000018.tif135168

[0105] These data show that for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2, when the mismatch is opposite rC, there is a large increase in mismatch discrimination (about 14.0 cycles and about 13.5 cycles, respectively). In particular, for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2, there is a large increase in mismatch discrimination for the rC:C pair (21.9 cycles and 15.2 cycles, respectively). When the mismatch is opposite rU, there is a significant increase in mismatch discrimination for Q48R SEL29 RNase H2 (about 11.2 cycles), but not for A107V SEL29 RNase H2 (about 4.3 cycles). A significant change in mismatch discrimination is seen for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 when the mismatch is opposite rA (about 8.5 cycles and about 6.0 cycles, respectively). When the mismatch is opposite rG, the change in mismatch discrimination is smaller; however, this lack of change in mismatch discrimination is less significant since mismatch discrimination using WT RNase H2 was already quite effective for rG. These increases in mismatch discrimination for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 are similar to the mismatch discrimination for background SEL29 RNase H2. Both mutations improve mismatch discrimination when the mismatch is placed directly opposite the RNA base, but to different extents and with different specificities.

[0106] Example 4: Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 increase mismatch discrimination compared to WT RNase H2 when mismatches are located on the 5' side of the RNA To determine the extent of mismatch discrimination using Q48R SEL29 RNase H2 enzyme and A107V SEL29 RNase H2 enzyme when the mismatch is located 5' to the RNA nucleotide, an assay was designed targeting rs113488022 (V600E SNP in the human BRAF gene) with the SNP located immediately 5' to the RNA. The primers used in this assay are shown in Table 10, SEQ ID NOs: 36-39. The assay was performed in a 10 μL reaction volume. Thermal cycling and data collection were performed on a CFX384® Real Time System (Bio-Rad®, Hercules, Calif.). Briefly, 200 nM (2 pmoles) of blocked forward primer (SEQ ID NO: 38 or 39) and 200 nM (2 pmoles) of unblocked reverse primer (SEQ ID NO: 37) or 200 nM (2 pmoles) of unblocked forward primer (SEQ ID NO: 36) and 200 nM (2 pmoles) of unblocked reverse primer (SEQ ID NO: 37) were mixed into 1× iQ™ SYBR® Green Supermix®. 5 mU or 10 mU of WT (SEQ ID NO: 1) Pa RNase 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. 2,000 copies of synthetic double-stranded gBlock® (IDT, Coralville, IA) template, each corresponding to a homozygous genotype at the rs113488022 SNP (SEQ ID NO: 40 or 41), were added to each reaction. Reactions were performed in triplicate. Reactions were run under the following conditions: 95°C, 100°F, 100°C, 100°F, 100°C, 150°F, 200°C, 250°C, 300°C, 500°F, 600°C, 700°C, 800°C, 900°C, 1000°C, 15 ... 3:00 →(95℃ 0:10 →60℃ 0:30 )×65. Fluorescence data for intercalated SYBR® Green was collected after each extension time point. After the assay was completed, the data was analyzed and the average Cq value and ΔC q Values ​​were calculated and the results are presented in Table 11.

[0107] [Table 10]

[0108] [Table 11] TIFF2024501832000021.tif99164

[0109] These data indicate that Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 significantly improve mismatch discrimination at the 5' side of the RNA nucleotide. q Quantification of ΔC increased from 3.8 cycles with WT PaRNase H2 to 5.9 cycles with A107V SEL29 RNase H2 (for 5 mU 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 5 mU of WT RNase H2 and 40 mU of Q48R SEL29 RNase H2). q Quantification of C increased from 10.1 cycles with WT PaRNase H2 to 11.2 cycles with A107V SEL29 RNase H2 (for 5 mU 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 5 mU of WT RNase H2 and 40 mU of Q48R SEL29 RNase H2). ΔC when using the WT enzyme qThe change in mismatch discrimination for the ArG primer is less significant since ArG primer was already quite effective for this primer. These increases in mismatch discrimination for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 are similar to the mismatch discrimination for background SEL29 RNase H2. Thus, Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 improve mismatch discrimination when the mismatch is placed 5' to the RNA base.

[0110] Example 5: Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 increase mismatch discrimination compared to WT RNase H2 when mismatches are located on the 3' side of the RNA To determine whether the Q48R SEL29 RNase H2 enzyme and the A107V SEL29 RNase H2 enzyme could improve mismatch discrimination when the mismatch was placed 3' to the RNA nucleotide, an assay was designed targeting the rs7583169 SNP and the rs3117947 SNP with a mismatch placed immediately 3' to the RNA. The primers used in this assay are shown in Table 12, SEQ ID NOs: 42-47. The assay was performed in a 10 μL reaction volume. Thermal cycling and data collection were performed on a CFX384® Real Time System (Bio-Rad®, Hercules, Calif.). Briefly, 200 nM (2 pmoles) of blocked forward primer (SEQ ID NO: 43 or 46) and 200 nM (2 pmoles) of unblocked reverse primer (SEQ ID NO: 44 or 47) or 200 nM (2 pmoles) of unblocked forward primer (SEQ ID NO: 42 or 45) and 200 nM (2 pmoles) of unblocked reverse primer (SEQ ID NO: 44 or 47) were mixed into 1x PrimeTime® Gene Expression Master Mix (IDT, Coralville, IA) with 3.0 mM (total concentration) MgCl2 and 0.5x EvaGreen® dye (Biotium Inc., Fremont, CA). 5mU or 10mU of WT (SEQ ID NO: 1) PaRNase H2 enzyme, 50mU or 100mU of Q48R SEL29 (SEQ ID NO: 18) RNase H2 enzyme, or 20mU or 40mU of A107V SEL29 (SEQ ID NO: 19) RNase H2 enzyme were added to each reaction. 20ng of cell line genomic DNA (cell lines NA12878 and NA24385; Coriell Institute for Medical Research, Camden, NJ) representing two homozygous genotypes at the rs7583169 SNP and rs3117947 SNP were added to each reaction. Reactions were performed in triplicate and results were averaged. Reactions were performed under the following conditions: 95°C, 37°C, 45°C, 50°C, 100°C, 1 ... 3:00 →(95℃0:10 →60℃ 0:30 )×65. Fluorescence data for the intercalated EvaGreen® was collected after each extension time point. After the assay was completed, the data was analyzed and the average C q value and ΔC q Values ​​were calculated and the results are presented in Table 13.

[0111] [Table 12]

[0112] [Table 13]

[0113] These data indicate that Q48R SEL29 and A107V SEL29 significantly increase mismatch discrimination at the 3' side of the RNA nucleotide. q Quantification of ΔC increased from 0.0 cycles with WT PaRNase H2 to 1.4 cycles with A107V SEL29 RNase H2 (for 5 mU of WT RNase H2 and 40 mU of A107V SEL29 RNase H2) and from 0.0 cycles with WT PaRNase H2 to 11.5 cycles with Q48R SEL29 RNase H2 (for 5 mU of WT RNase H2 and 100 mU of Q48R SEL29 RNase H2). qQuantification of mismatch discrimination increased from 10.7 cycles with WT PaRNase H2 to 22.3 cycles with A107V SEL29 RNase H2 (for 5 mU of WT RNase H2 and 40 mU of A107V SEL29 RNase H2) and from 10.7 cycles with WT PaRNase H2 to 20.4 cycles with Q48R SEL29 RNase H2 (for 5 mU of WT RNase H2 and 100 mU of Q48R SEL29 RNase H2). These increases in mismatch discrimination for Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 are significantly greater than the mismatch discrimination for background SEL29 RNase H2. Thus, Q48R SEL29 RNase H2 and A107V SEL29 RNase H2 improve mismatch discrimination when the mismatch is located 3' to the RNA base.

[0114] Example 6: Use of Q48R SEL29 Hybrid RNase H2 Protein and A107V SEL29 Hybrid RNase H2 Protein in LAMP Reaction This example outlines methods demonstrating the use of a hybrid RNase H2 protein to reduce primer-dimer formation in LAMP reactions.

[0115] To assess the functionality of rhPrimers for LAMP protocols involving different RNase H2 proteins (WT RNase H2 protein or hybrid RNase H2 protein), three assays could be designed using (1) unmodified control primers; rDDDDMx primers [in the sequence, "r" is an RNA base, "D" is a DNA base, "m" is a mismatch, and "x" is a C3 spacer], "Gen1"; and rDxxDM primers, "Gen2". The details of the assays used are detailed in Tables 14, 15, and 16. LAMP reactions utilizing each type of primer are held at 25°C (room temperature) for 0 or 2 hours before testing. This allows for the formation of primer dimer products. After the room temperature hold, all reactions are performed at 65°C for 2 hours in a BioRad CFX384 or Roche LightCycler 480. Signal development in all of these reactions is performed 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] TIFF2024501832000026.tif116164

[0118] [Table 16] TIFF2024501832000028.tif65164

[0119] Each assay may be performed as follows: Samples are Coriell gDNA or lambda phage genomic DNA. Each assay condition may be run in triplicate with sample inputs of 5 ng lambda phage genomic DNA or 20 ng human genomic DNA. For each assay, reactions may be run using unmodified primer intercalating dye (e.g., EvaGreen) and cleavable blocked primers with intercalating dye. For each assay, reactions are run using zero or titrated levels of RNase H2 (WT RNase H2 protein or hybrid RNase H2 protein).

[0120] Each assay is run in triplicate. Comparison of the unmodified and modified LAMP assays is performed by comparing the length of time required for the formation of signal generating products. Hybrid RNase H2 protein in LAMP reactions is expected to produce these products significantly later than reactions containing conventional wild-type RNase H2 protein.

[0121] 25 μL of EvaGreen reaction mixture 12.5 μL of 2x Master Mix (25°C, pH 8.8, 1x 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 of EvaGreen dye 0.2 μM F3 primer 0.2 μM B3 primer 8U of BST DNA polymerase (New England Biolabs: https: / / www.neb.com / product / m0275-bst-dna-polymerase-large-fragment) 1 μL of Hybrid RNase H2 Protein (for RNase H2 control, no buffer D is used) Up to 25uL nuclease-free water 2 μL of sample (10 ng / μL human gDNA or 2.5 ng / uL lambda genomic DNA) Includes.

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

[0123] Each step is followed by two PCR cycling steps with SPRI (Solid Phase Reversible Immobilization, Beckman Coulter Life sciences, Indianapolis, IN) cleanup. The purpose of the first PCR step (PCR1) is to incorporate a 6-nucleotide degenerate UMI on each side of the target amplicon. This step also involves the use of a 3' blocked primer, requiring RNase H2 to cleave the protector, allowing high fidelity DNA polymerase to extend and amplify each target. The target-specific assay panel contains 177 proprietary primer pairs, resulting in approximately 20% of the total reads being primer dimers when wild-type PaRNase H2 is present in PCR1. This panel is ideal for investigating Q48R SEL29 RNase H2, as it results in a large amount of primer dimers.

[0124] Various 10-fold titrations of Q48R SEL29 RNase H2 and wild-type Pa RNase 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 was in a final volume of 20 μL and contained 10 nM (200 femtomoles) of each forward and reverse primer, 0.03 U / μL 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 a final concentration of 1× RNase H2 in proprietary high fidelity polymerase buffer. Thermal cycling was performed on a T100 Thermal Cycler (Bio-Rad®, Hercules, CA) with the cycling conditions listed in Table 17. PCR1 thermal cycling was immediately followed by SPRI cleanup. 1.25x (25 μL) of AMPure magnetic beads (Beckman Coulter Life Sciences, Indianapolis, IN) was added to each well and mixed thoroughly. The plate was incubated for 5 min at room temperature on the bench, followed by 5 min on the magnet. The supernatant was discarded and the library was washed twice with 80% ethanol. Samples were eluted in 22 μL of IDTE pH 7.5 (IDT, Coralville, IA). 20 μL of the eluted product was carried over 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. PCR2 reactions were performed in a final volume of 50 μL. A 2x version of the high fidelity polymerase buffer used in PCR1 was added to each eluted sample from PCR1 to achieve a final concentration of 1x. Each reaction 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) with the cycling conditions listed in Table 18. Immediately after PCR2, a SPRI cleanup was performed, which is identical to the method listed above, except for the AMPure bead concentration used at 0.9x after PCR2. Samples were eluted in 22 μL of IDTE pH 7.5 (IDT, Coralville, Iowa). 20 μL of the elution product was taken and stored at −20° C. until sequencing.

[0127] [Table 18]

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

[0129] For the purposes of this disclosure, the following result calculations and terminology are listed here: The amount of primer dimers resulting during amplification of a sequencing library is defined by the dimer rate. 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 pass the cf (chastity filter)). The mapping rate is calculated by taking the total number of mapped reads and dividing by the number of QC-passing reads. The on-target rate is calculated by taking the total number of on-target reads and dividing by the number of QC-passing reads. The total amplicon uniformity or ≧0.2-fold amplicon uniformity is calculated as the percentage of normal amplicons that are equal to or greater than 0.2-fold the average amplicon coverage. The dropout rate or ≦0.05-fold uniformity is calculated as the percentage of normal amplicons that are less than 0.05-fold the average amplicon coverage. The uniformity distribution of amplicons is calculated by comparing the coverage of each amplicon relative to the average amplicon coverage for a given sample and plotting within the following ranges: 0.1-0.2x, 0.2-0.5x, 0.5-1.5x, 1.5-2.5x, and 2.5-5x.

[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 each primer dimer contributes to the total dimer rate, and is calculated using the following formula:

[0131]

number

[0132] For all concentrations tested, Q48R SEL29 RNase H2 reduced dimer rates compared to wild-type PaRNase H2 and also increased mapping and on-target rates. At the lowest concentration (8.75 mU / μL), Q48R SEL29 RNase H2 produced libraries with 4% dimers compared to control wild-type PaRNase H2, which produced 11% dimers (Figure 1, panel A).

[0133] The mapping and on-target rates for the library generated 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] Dimer rate is divided into individual primer dimers resulting, and then normalized to determine the contribution of each primer dimer to the total dimer rate described above (normalized dimer count).Q48R SEL29 RNase H2 reduces the amount of most primer dimers by half.For example, the top dimer (NOTCH1_19.GAP95.9623.relax.t0_FOR:NOTCH1_9.GAP104.1246.relax.t0_REV) identified in all sample libraries of the library made with the lowest concentration of Q48R Mut 29 RNase H2 (8.75mU / μL) only accounts for 1.23% of the total dimer rate, compared to 2.83% for wild-type PaRNase H2. In the case of the undetermined:TERT_15.GAP27.511.relax.t0_REV Q48R SEL29 RNase H2 library, some dimers were reduced in number, resulting in 0.11% dimers, which is 75% less than the wild-type PaRNase H2, but in this case this dimer contributed 0.47% to the total dimer rate (Table 19 and Figure 2).

[0135] [Table 19] TIFF2024501832000033.tif225165TIFF2024501832000034.tif57161

[0136] The ability of Q48R SEL29 RNase H2 to reduce dimer rate does not affect the metrics of library yield, total uniformity, and dropout rate or uniformity distribution. Q48R SEL29 RNase H2 results in similar library yields compared to wild-type Pa RNase H2 enzyme at all titration concentrations tested, as evidenced by the average amplicon coverage (Figure 3, Panel A). In addition, total amplicon uniformity of ≥ 0.2-fold and amplicon dropout rate (amplicon uniformity of ≤ 0.05-fold) are comparable between Q48R SEL29 RNase H2 and wild-type Pa enzyme at all titration concentrations tested (Figure 3, Panels B and C). Furthermore, the uniformity distribution between Q48R SEL29 and wild-type Pa RNase H2 appears to be similar (Figure 4). Q48R SEL29 RNase H2 reduces primer-dimer formation during library generation in the rhAmpseq workflow compared to standard wild-type PaRNase H2, without altering other important sequencing metrics.

[0137] Taken together, these data indicate that Q48R SEL29 RNase H2 improves the generation of multiplexed next-generation sequencing libraries using high-fidelity DNA polymerases in high-fidelity buffers.

[0138] Example 8: Exemplary amino acid and nucleic acid sequences encoding RNase H2 proteins Exemplary amino acid and nucleic acid sequences encoding RNase H2 proteins are provided below.

[0139] [Table 20] TIFF2024501832000036.tif250161TIFF2024501832000037.tif251161TIFF2024501832000038.tif249161TIFF2024501832000039.tif250161TIFF2024501832000040.tif145161

[0140]

Table 21

[0141]

Table 22

[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 in this specification, including publications, patent applications, and patents, are herein incorporated by reference in their entireties to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth herein.

[0144] Preferred embodiments of the present invention are described herein, including the best mode for carrying out the present invention known to the inventors. Variations to these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that those skilled in the art will utilize such variations as necessary, and the inventors intend for the invention to be carried out in ways other than those specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the elements described above in all possible variations thereof is also encompassed by the present invention, unless otherwise indicated herein or the context clearly indicates to the contrary.

Claims

1. The organism Pyrococcus abyssi (P a.), Thermococcus kodakarensis (T. kod) and Pyrococcus furiosus, comprising a fragment of the amino acid sequence: amino acid residues 26-40 and residues 100-120 of T. kod RNase H2; SEQ ID NOs: 2 and 3; and SEQ ID NOs:14-20; A hybrid RNase H2 protein comprising one member selected from the group consisting of:

2. A recombinant nucleic acid encoding any of the hybrid RNase H2 proteins of claim 1.

3. 11. A method for performing primer extension, comprising contacting the hybrid RNase H2 protein of claim 1 with a primer, a polynucleotide template, nucleoside triphosphates and a DNA polymerase under conditions suitable for primer extension, thereby generating an extended primer.

4. 4. The method of claim 3, wherein the DNA polymerase comprises a high-fidelity archaeal DNA polymerase, the primer comprises a blocked cleavable primer, and the primer extension method comprises a method for performing a polymerase chain reaction (PCR) and improving mismatch discrimination on the 3' side in a primer:polynucleotide hybrid formed between the primer and a polynucleotide template.

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

6. 11. A method for performing rhPCR, comprising the steps of performing primer extension with a hybrid RNase H2 of claim 1, a high fidelity archaeal DNA polymerase and a blocked cleavable primer comprising a cleavage domain and a blocking group attached to the 5' side of the 3' terminal residue, which prevents the primer from being used as a template for DNA synthesis, wherein the hybrid RNase H2 enzyme can be reversibly inactivated by chemical modification, an aptamer or a blocking antibody.

7. 7. The method of claim 6, wherein the blocking group is selected from the group consisting of one or more abasic residues or one member selected from the group consisting of RDDDDx, RDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD, where R is an RNA residue, D is a DNA residue, M is a mismatch residue and x is a C3 spacer or other method of blocking DNA.

8. 7. The method of claim 6, wherein the blocking group comprises a label that allows for detection of the extension amplification reaction, the label being attached to the 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 may comprise any of the following moieties: a DNA residue, a single RNA residue, two adjacent RNA residues, a contiguous sequence of three or more RNA residues, an abasic residue, one or more 2′ modified nucleosides, an internucleotide modified phosphate linkage, The method of claim 6 , comprising one or more of:

10. 7. The method of claim 6, wherein the cleavage domain comprises one or more 2'-modified nucleosides selected from the group consisting of 2'-O-alkyl RNA nucleosides, 2'-O-methyl RNA nucleosides, 2'-fluoro nucleosides, locked nucleic acids, 2'-ethylene nucleic acid residues, 2'-alkyl nucleosides, 2'-amino nucleosides, and 2'-thionucleosides.

11. 1. A method for amplifying a target DNA sequence, comprising: (a)(i) a blocked cleavable primer having a cleavage domain that is cleavable by an RNase H2 enzyme located 5' to a blocking group attached to the 5' side of the 3' terminal residue of the blocked cleavable primer, wherein the blocking group prevents primer extension and / or inhibits the blocked cleavable primer from being used as a template for DNA synthesis; (ii) a sample nucleic acid, which may or may not be a target sequence; (iii) a high-fidelity archaeal DNA polymerase; and (iv) the hybrid RNase H2 protein of claim 1, which is capable of being reversibly inactivated by chemical modification, an aptamer, or a blocking antibody. providing a reaction mixture comprising: (b) hybridizing the blocked cleavable primer to the target DNA sequence to form a double-stranded substrate; (c) cleaving the hybridized blocked cleavable primer with a hybrid RNase H2 enzyme at a cleavage site within or adjacent to the cleavage domain to remove the blocking group from the blocked cleavable primer. The method includes:

12. 12. The method of claim 11, wherein the blocking group is selected from the group consisting of: one or more abasic residues and one member selected from the group consisting of RDDDDx, RDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD, where R is an RNA residue, D is a DNA residue, M is a mismatch residue, and x is a C3 spacer or other method of blocking extension by DNA polymerase.

13. 12. The method of claim 11, wherein the blocking group comprises a label that allows for detection of the extension amplification reaction, the label being attached to the blocked cleavable primer 3' from the cleavage site, and the label is a fluorophore or a mass tag.

14. 12. The method of claim 11, wherein the cleavage domain comprises one or more of the following moieties: a DNA residue, a single RNA residue, two adjacent RNA residues, a consecutive sequence of three or more RNA residues, an abasic residue, one or more 2'-modified nucleosides, or internucleotide-modified phosphate linkages.

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

16. 11. A kit for making an extension primer, comprising at least one container providing a hybrid RNase H2 protein of claim 1 and one or more additional containers selected from the group consisting of: (a) a container providing a primer capable of hybridizing to a predetermined polynucleotide template under primer extension conditions; (b) a container comprising one or more blocked cleavable primers; (c) a container providing nucleoside triphosphates; (d) a container providing a buffer suitable for primer extension, and (e) a container providing a high fidelity archaeal DNA polymerase.

17. 13. A kit for performing amplification of a target DNA sequence, comprising a reaction buffer containing the RNase H2 of claim 1 and a high-fidelity archaeal DNA polymerase.

18. 18. The kit of claim 17, further comprising one or more oligonucleotide primers, at least one of which comprises a blocked cleavable primer having a cleavage domain that is cleavable by an RNase H2 enzyme and a blocking group located 5' to a blocking group linked at or near the 3' end of the blocked cleavable primer, wherein the blocking group prevents primer extension and / or inhibits the oligonucleotide primer from functioning as a template for DNA synthesis.

19. 20. The kit of claim 18, wherein the blocking group comprises one member selected from the group consisting of RDDDDx, RDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD, where R is an RNA residue, D is a DNA residue, M is a mismatch residue and x is a C3 spacer or other moiety that blocks extension by DNA polymerase.

20. 1. A method for preparing an amplification product library of a template nucleic acid, comprising: Forming a mixture, the mixture comprising: A population of nucleic acids; at least one blocked cleavable primer; A hybrid RNase H2 protein of SEQ ID NO: 18; dNTPs; A high-fidelity archaeal DNA polymerase; and High fidelity archaeal DNA polymerase buffer Including, forming a hybrid duplex between at least one blocked cleavable primer and the population of nucleic acids in the mixture; cleaving at least one blocked cleavable primer with a hybrid mutant RNase H2 protein to generate at least one active primer capable of primer extension by a DNA polymerase; and extending at least one active primer with a DNA polymerase in a buffer solution under conditions that permit amplification of one or more template nucleic acids from the population of nucleic acids, thereby generating an amplification product of the template nucleic acid. The method includes:

21. 1. A method for performing massively parallel sequencing, comprising: A population of nucleic acids; A hybrid RNase H2 mutant protein of SEQ ID NO: 18; at least one blocked cleavable primer; DNA polymerase; dNTPs; and buffer solution preparing a library population of template nucleic acids, comprising performing PCR with a mixture comprising: ;and Sequencing a plurality of desired template nucleic acids from the library population of template nucleic acids. The method includes:

22. 1. A method for detecting a SNP-containing nucleic acid template from a library of amplification products of a nucleic acid template, comprising: Forming a mixture, the mixture comprising: A library of amplification products of a nucleic acid template; at least one blocked cleavable primer; A hybrid RNase H2 protein of SEQ ID NO: 18; dNTPs; DNA polymerase; and buffer solution Including, forming a hybrid duplex between at least one blocked cleavable primer in the mixture and a SNP-containing nucleic acid template within the amplification product library of nucleic acid templates; cleaving at least one blocked cleavable primer of the hybrid duplex with a hybrid RNase H2 protein to generate at least one active primer capable of primer extension of the hybrid duplex by a DNA polymerase; and extending at least one active primer in the hybrid duplex with a DNA polymerase in a buffer solution under conditions that allow amplification of one or more template nucleic acids from the library of amplification products of the nucleic acid templates, thereby detecting the SNP-containing nucleic acid templates. The method includes:

23. 1. A method for performing a loop-mediated amplification reaction, comprising: Forming a mixture, the mixture comprising: A nucleic acid template; four blocked cleavable primers which form a duplex with a nucleic acid template which is a substrate for RNase H2 protein; RNase H2 protein of SEQ ID NO: 18; DNA polymerase protein; dNTPs; and buffer solution and subjecting the mixture to isothermal amplification cycles. The method includes:

24. 24. A mixture product produced using the method of any one of claims 3, 6, 11, 20, 21, 22 and 23 with an RNase H2 of SEQ ID NO: 18, wherein the mixture product produced from the RNase H2 of SEQ ID NO: 18 comprises a reduced population of primer dimer molecular species compared to the mixture product produced with a wild type P.a. RNase H2 of SEQ ID NO:

1.

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

1.

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

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