CN116829720A

CN116829720A - Primer dimer reduction and off-target amplified RNase H2mutants in RHPCR-based amplicon sequencing using high fidelity DNA polymerase

Info

Publication number: CN116829720A
Application number: CN202180087457.9A
Authority: CN
Inventors: J·多博斯; J·弗勒里格; K·佩尔施巴彻; K·贝尔兹; S·罗斯; M·A·贝尔克
Original assignee: Integrated DNA Technologies Inc
Current assignee: Integrated DNA Technologies Inc
Priority date: 2020-12-24
Filing date: 2021-12-22
Publication date: 2023-09-29

Abstract

The present application relates to hybrid rnase H2 proteins, including fragments of amino acid sequences from deep-sea thermococcus (p.a.), rhodococcus kochianus (t.kod) and pyrococcus intensive organisms, and methods of using the same to improve mismatch discrimination and activity in high-fidelity DNA polymerase buffers.

Description

Primer dimer reduction and off-target amplified RNase H2mutants in RHPCR-based amplicon sequencing using high fidelity DNA polymerase

Cross Reference to Related Applications

The present application is based on the priority of U.S. patent application Ser. No. 63/130,548 entitled "RNASE H2MUTANTS THAT ENHANCE MISMATCH DICRIMINATION AND ACTIVITY IN HIGH-FIDELITY POLYMERASE BUFFER" filed on 12/24/2020, and U.S. patent application Ser. No. 63/277,273 entitled "RNASE H2MUTANTS THAT REDUCE PRIMER DIMERS AND OFF-TARGET AMPLIFICATION IN RHPCR-BASED AMPLICON SEQUENCING WITH HIGH-FIDELITY DNA POLYMERASES" filed on 11/2021, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to type II rnase H (hereinafter rnase H2) hybrid enzyme variants and methods of cleaving nucleic acid strands to initiate, assist, monitor or perform a bioassay.

Background

The rnase H2 enzyme family has been extensively characterized. These enzymes have substrate specificity for cleavage of a single ribonucleotide that is inserted into a DNA sequence (duplex form) (Eder, et al, (1993) Biochimie,75, 123-126). Interestingly, cleavage occurred 5' to the RNA residue (see scheme I). These enzymes, their properties and their use in bioassays are summarized in Walder et al, U.S. Pat. No. 8,911,948B2.

RNase H2 enzyme isolated from Thermococcus furiosus (Pyrococcus abyssi) (P.a.) cleaves 5' of ribonucleotides that are embedded in a nucleic acid strand that otherwise consists entirely of DNA. RNase H2-dependent PCR (rhPCR) improves the specificity of PCR, in which primers containing a single ribonucleotide near the 3 'terminus are cleaved using thermostable RNase H2 enzyme, removing the blocking group at the 3' terminus (Dobosy et al, 2011; U.S. Pat. No. 8,911,948B2). The primer cannot be extended initially by a DNA polymerase, but after the rnase H2 removes the blocking group from the 3' end, the primer can be extended. Rnase H2 is sensitive to single base mismatches in DNA-RNA heteroduplex near ribonucleotides and cleaves templates containing mismatches at a greatly reduced rate, which allows perfectly matched duplex to be preferentially cleaved and extended. This results in increased specificity of the rhPCR reaction and reduced primer dimer formation and other off-target amplification.

Although rhPCR enhances the specificity of PCR, it is currently limited. The obvious mismatch discrimination of rhPCR is lower than what should theoretically be achieved. WT p.a. rnase H2 recognizes to a large extent single base mismatches directly opposite the RNA bases, but the efficiency varies depending on the nature of the mismatch. Furthermore, the mismatch recognition of native enzymes at the direct 5 'or 3' positions of RNA bases is relatively limited. Despite this limitation, it may be advantageous to place mutations at these positions. For example, in PCR amplification after primer cleavage, a direct 5' mismatch of RNA can be used as a secondary selection step, wherein a discriminating DNA polymerase (e.g., H784Q Thermus aquaticus (Thermus aquaticus) DNA polymerase) is used (see U.S. patent application Ser. No. 15/361280). Placing a mismatch at the 3' of the RNA reduces the likelihood of template switching, as mismatch recognition occurs in each cycle, rather than once during template switching.

Despite the utility of rhPCR, mismatch discrimination of wild-type p.a.rnase H2 may "bleed" resulting in some amplification of primer dimers. Primer dimers generated during amplification of the sequencing library are problematic in that they can bind to the Illumina flow cell and be sequenced, but do not provide any meaningful data. High levels of primer dimer will reduce the proportion of reads mapped to the target of interest, ultimately reducing assay sensitivity or requiring a significant increase in sequencing cost to generate the number of mid-target reads required to detect low frequency variation. Low frequency variation detection is also affected by amplification errors introduced during PCR. The use of high fidelity DNA polymerase and optimized buffer conditions can reduce error rates. However, these buffer conditions reduced the enzymatic activity and mismatch sensitivity of wild-type p.a.rnase H2 in rhPCR.

The use of rnase H2mutants produced by partial recombination of the amino acid sequence of wild-type p.a. rnase H2 with sequences from other related species has been previously shown to result in improved rnase H2 enzyme activity in high fidelity DNA polymerase buffers. Two of these mutants, Q48RSEL29 rnase H2 and Al07V SEL29 rnase H2, showed improved enzymatic activity when using a KOD DNA polymerase reaction buffer. Furthermore, both mutants showed enhanced mismatch discrimination relative to the RNA base and 3 'and 5' mismatches of the RNA base compared to wild-type P.a rnase H2. See U.S. provisional patent application Ser. No. 63/130,548, entitled "RNASE H2MUTANTS THAT ENHANCE MISMATCH DICRIMINATION AND ACTIVITY IN HIGH-FIDELITY POLYMERASE BUFFER" (attorney docket No. IDT 01-018-PRO), filed on month 12, 24, 2020, the contents of which are incorporated by reference in their entirety.

The present disclosure relates to one of these novel hybrid rnase H2 enzyme variants, Q48R SEL29 rnase H2, in a multiplex rhamp seq workflow comprising high fidelity DNA polymerase and buffer. Q48R SEL29 RNase H2 reduced primer dimer generated during PCR amplification compared to wild-type P.a.RNase H2, thereby improving mapping rate and mid-target rate. While improving these metrics, Q48RSEL29 rnase H2 had no effect on other key sequencing metrics, including amplicon uniformity, amplicon loss rate, and amplicon uniformity distribution.

Disclosure of Invention

In a first aspect, a hybrid rnase H2 protein is provided. Hybrid rnase H2 proteins include amino acid sequence fragments from the organisms pyrococcus abyssal (Pyrococcus abyssi) (p.a.), pyrococcus costa (t.kod) and pyrococcus intensiviss (Pyrococcus furiosus).

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

In a third aspect, a method of performing primer extension is provided. The method comprises the following steps: contacting a hybrid rnase H2 protein disclosed herein with a primer, a polynucleotide template, nucleoside triphosphates, and a DNA polymerase under conditions suitable for a primer extension method, thereby producing an extended primer.

In a fourth aspect, a reaction mixture is provided. The reaction mixture includes the hybrid rnase H2 protein described herein, at least one primer, a polynucleotide template, nucleoside triphosphates, and a DNA polymerase.

In a fifth aspect, a method for performing rhPCR is provided. The method comprises the step of primer extension with the hybrid rnase H2, DNA polymerase and primers described herein.

In a sixth aspect, a method of amplifying a target DNA sequence is provided. The method comprises several steps. The first step is to provide a reaction mixture comprising: (i) An oligonucleotide primer having a cleavage domain cleavable by an rnase H2 enzyme, located 5 'of a blocking group attached at or near the 3' end of the oligonucleotide primer, wherein the blocking group prevents primer extension and/or inhibits oligonucleotide primer as a template for DNA synthesis; (ii) a sample nucleic acid which may or may not be a target sequence; (iii) A DNA polymerase, and (iv) a hybrid rnase H2 protein disclosed herein. The second step includes hybridizing the oligonucleotide primer to 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 seventh aspect, a kit for generating an extended primer is provided. The kit comprises at least one container that provides a hybrid rnase H2 protein disclosed herein.

In an eighth aspect, a kit for performing amplification of a target DNA sequence is provided. The kit includes a reaction buffer comprising rnase H2 and a high fidelity archaea DNA polymerase as described herein.

In a ninth aspect, a method of preparing a library of template nucleic acid amplicons is provided. The method comprises several steps. The first step is to form a mixture comprising a population of nucleic acids, at least a blocked cleavable primer, a hybrid rnase H2 protein, dntps, a DNA polymerase, and a buffer, such that hybridization duplex is formed between at least the blocked cleavable primer and the population of nucleic acids in the mixture. The second step is cleaving the at least one blocked cleavable primer with a hybrid rnase H2 protein to produce 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 in the buffer with the DNA polymerase under conditions that allow for amplification of one or more template nucleic acids from the population of nucleic acids, thereby generating amplicons of the template nucleic acids. In a first aspect, the hybrid RNase H2 protein is selected from Q48R SEL29 (SEQ ID NO: 18) or others. In a second aspect, the DNA polymerase is KOD DNA polymerase, or other high fidelity archaea DNA polymerase. In a third aspect, the buffer is a high fidelity archaea DNA polymerase buffer.

In a tenth aspect, a method of performing massively parallel sequencing is provided. The method comprises several steps. The first step is to prepare a template nucleic acid library population using a nucleic acid population, a hybrid rnase H2 mutein, 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 template nucleic acid library population. In a first aspect, the hybrid RNase H2 protein is selected from Q48R SEL29 (SEQ ID NO: 18) or others.

In an eleventh aspect, there is provided a method of detecting a SNP containing nucleic acid template from a nucleic acid template amplicon library, the method comprising: the method comprises several steps. The first step includes forming a mixture comprising a library of nucleic acid template amplicons; at least one blocked cleavable primer; hybrid mutant rnase H2 protein; dNTP; a DNA polymerase; and a buffer. Hybridization duplex is formed between at least the blocked cleavable primer and the SNP containing nucleic acid template in the nucleic acid template amplicon library in the mixture. The second step comprises cleaving at least one blocked cleavable primer of the hybridization duplex with the hybrid rnase H2 protein to generate at least one active primer capable of primer extension of the hybridization duplex by a DNA polymerase. The third step comprises extending at least one active primer in the duplex with a DNA polymerase in a buffer under conditions that allow amplification of one or more template nucleic acids from the nucleic acid template amplicon library, thereby detecting a nucleic acid template comprising a SNP. In a first aspect, the method comprises a hybridization mutant RNase H2 protein selected from Q48R SEL29 (SEQ ID NO: 18) or others. In a second aspect, the method comprises a buffer that is a high fidelity archaea DNA polymerase buffer.

In a twelfth aspect, a method of performing a loop-mediated amplification reaction is provided. The method comprises two steps. The first step includes forming a mixture comprising nucleic acid templates; four blocked cleavable primers, wherein the blocked cleavable primers form a duplex with a nucleic acid template that is a substrate for an rnase H2 protein; an rnase H2 protein, wherein the rnase H2 protein is selected from Q48R SEL29 (SEQ id No.: 18) or others; a DNA polymerase protein; dNTP; and a buffer. The second step involves performing an isothermal amplification cycle with the mixture.

In a thirteenth aspect, a method of performing an rhPCR assay with reduced primer dimer formation is provided. The method includes primer extension with Q48R SEL29 RNase H2 (SEQ ID NO: 18). The reduced primer dimer formation corresponds to the amount of primer dimer that was reduced during the rhPCR assay with Q48R SEL29 rnase H2 (SEQ ID No.: 18) when compared to the rhPCR assay with wild-type p.a. rnase H2 (SEQ ID No.: 1).

In a fourteenth aspect, a method of rhPCR assay is provided having improved mapping and targeting rates for desired products. The method includes primer extension with Q48R SEL29 RNase H2 (SEQ ID NO: 18). The improved mapping and mid-target rates correspond to increased mapping and mid-target amplification of the desired product formed during the rhPCR assay with Q48R SEL29 rnase H2 (SEQ ID No.: 18) when compared to the rhPCR assay with wild-type p.a. rnase H2 (SEQ ID No.: 1).

Drawings

Fig. 1 depicts the following: an exemplary graph showing that dimer rates were reduced at all enzyme concentrations using mutant Q48R SEL29 rnase H2 enzyme compared to wild-type p.a. rnase H2 enzyme (panel a); exemplary data, which demonstrate that mapping rates are higher at all rnase H2 concentrations when using mutant Q48R SEL29 rnase H2 enzyme compared to wild-type p.a. rnase H2 enzyme (panel B); exemplary data, which demonstrate that the target rate is higher at all enzyme concentrations when using mutant Q48R SEL29 rnase H2 enzyme compared to wild-type p.a. rnase H2 enzyme (panel C).

FIG. 2 depicts an example of the average normalized dimer counts for each identified primer pair, wherein the mutant Q48R SEL29 RNase H2 enzyme halved the majority of primer dimers identified as compared to the wild-type P.a. RNase H2 enzyme.

FIG. 3 depicts exemplary data amplicon uniformity ≡0.2X and amplicon uniformity ≡0.05X (discard rate), where library yields were comparable between mutant Q48R SEL29 RNase H2 enzyme and wild-type P.a.RNase H2 enzyme at all concentrations (panel A); at the different concentrations tested, the overall amplicon uniformity of mutant Q48R SEL29 rnase H2 enzyme was ≡0.2X comparable to wild-type p.a. rnase H2 enzyme (panel B); and amplicon loss rates were similar between mutant Q48R SEL29 rnase H2 enzyme and wild-type p.a. rnase H2 enzyme at all titration concentrations tested (panel C).

FIG. 4 depicts exemplary data showing that uniformity profiles (percentage of amplicons with coverage of 0-0.1X, 0.1-0.2X, 0.2X-0.5X, 0.5X-1.5X, 1.5X-2.5X, and 2.5-5X compared to the average coverage of amplicons) are comparable between mutant Q48R SEL29 RNase H2 enzyme and wild-type P.a.RNase H2 enzyme.

Detailed Description

The present invention provides novel hybrid rnase H2 enzyme variants that enhance enzyme activity during rhPCR using certain DNA polymerase buffers while retaining their ability to retain or enhance mismatch discrimination in duplex templates. Rnase H2 enzyme hybrids combine amino acid sequence fragments from the organisms pyrococcus abyssal (Pyrococcus abyssi) (p.a.), pyrococcus kodakodii (t.kod) and pyrococcus furiosus (Pyrococcus furiosus). The hybrid rnase H2 enzyme thus produced and mutants selected based on these enzymes significantly enhance mismatch discrimination. In particular, in methods of performing primer extension, performing rhPCR, amplifying a target DNA sequence, performing large-scale parallel sequencing, detecting a SNP-containing nucleic acid template from a nucleic acid template amplicon library, and performing a loop-mediated amplification reaction, Q48R SEL29 RNase H2 (SEQ ID NO: 18) shows that the resulting product mixture has a reduced population of primer dimer species relative to the product mixture produced with wild-type P.a.RNase H2 (SEQ ID NO: 1).

Definition of the definition

To aid in understanding the invention, several terms are defined below.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Unless otherwise described, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"). Recitation of ranges of values herein are merely intended to serve 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 the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

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-glycosidic purine or pyrimidine base. There is no intended distinction in length between the terms "nucleic acid", "oligonucleotide" and "polynucleotide", and these terms will be 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, the oligonucleotides may also comprise nucleotide analogs in which the base, sugar or phosphate backbone is modified, as well as non-purine or non-pyrimidine nucleotide analogs.

Oligonucleotides may be prepared by any suitable method, including direct chemical synthesis by a method such as the phosphotriester method of Narag et al 1979, meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al 1979, meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Brown et al 1981,Tetrahedron Lett.22:1859-1862; and U.S. Pat. No. 4,458,066, each incorporated herein by reference. An overview of the methods of synthesis of conjugates of oligonucleotides and modified nucleotides is provided in Goodchild,1990,Bioconjugate Chemistry 1 (3): 165-187, which is incorporated herein by reference.

As used herein, the term "primer" refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. These conditions include inducing synthesis of primer extension products complementary to the nucleic acid strand in the presence of four different nucleoside triphosphates and an extender (e.g., a DNA polymerase or reverse transcriptase) in an appropriate buffer at an appropriate temperature. Primer extension may also be performed in the absence of one or more nucleoside triphosphates, in which case limited length extension products are produced. As used herein, the term "primer" is intended to include oligonucleotides used in ligation-mediated reactions, wherein one oligonucleotide is "extended" by ligation to a second oligonucleotide hybridized at an adjacent location. Thus, the term "primer extension" as used herein refers to both the polymerization of a single nucleoside triphosphate using a primer as a starting point for DNA synthesis and the ligation of two oligonucleotides to form an extension product.

The primer is preferably single stranded DNA. The appropriate length of the primer depends on the intended use of the primer, but is generally in the range of 6 to 50 nucleotides, preferably 15-35 nucleotides. Short primer molecules typically require lower temperatures to form sufficiently stable hybridization complexes with the template. The primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize to the template. The design of suitable primers for amplifying a given target sequence is well known in the art and described in the literature cited herein.

The primer may comprise additional features that allow detection or immobilization of the primer without altering the basic properties of the primer (i.e., the properties that act as starting points for DNA synthesis). For example, the primer may contain an additional nucleic acid sequence at the 5' end that does not hybridize to the target nucleic acid, but facilitates cloning or detection of the amplified product. The region of the primer that is sufficiently complementary to the template to hybridize is referred to herein as the hybridization region.

The terms "target," "target sequence," "target region," and "target nucleic acid" as used herein are synonymous and refer to a region or sequence of nucleic acid to be amplified, sequenced, or detected.

The term "hybridization" as used herein refers to the formation of a duplex structure from two single stranded nucleic acids due to complementary base pairing. Hybridization may occur between perfectly complementary nucleic acid strands or between "substantially complementary" nucleic acid strands that contain small mismatch regions. Conditions under which perfectly complementary nucleic acid strands hybridize are strongly preferred as "stringent hybridization conditions" or "sequence-specific hybridization conditions". Under less stringent hybridization conditions, stable duplex of substantially complementary sequences can be obtained; the degree of tolerable mismatch can be controlled by appropriate adjustment of hybridization conditions. One skilled in the art of nucleic acid technology can empirically determine double strand stability, wherein a number of variables are considered, including, for example, the length and base pair composition of the oligonucleotide, the ionic strength, and the incidence of mismatched base pairs, following guidelines in the art (see, e.g., sambrook et al, 1989,Molecular Cloning-A Laboratory Manual, cold Spring Harbor Laboratory, cold Spring Harbor, new York; wetdur, 1991,Critical Review in Biochem.and Mol.Biol.26 (3/4): 227-259; and Owczarzy et al, 2008, biochemistry,47:5336-5353, incorporated herein by reference).

The term "amplification reaction" refers to any chemical reaction, including enzymatic reactions, that results in increased copies of a template nucleic acid sequence or 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;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" generally include two or three-step cycles. The two-step cycle has a high temperature denaturation step followed by a hybridization/extension (or ligation) step. The three-step cycle includes a denaturation step, followed by a hybridization step, and then a separate extension or ligation step.

As used herein, "polymerase" refers to an enzyme that catalyzes the polymerization of nucleotides. Typically, the enzyme will initiate synthesis at the 3' end of the primer that anneals to the nucleic acid template sequence. "DNA polymerase" catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, the Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al, 1991, gene, 108:1), the E.coli DNA polymerase I (Lecote and Doubleday,1983,Nucleic Acids Res.11:7505), the T7 DNA polymerase (Nordstrom et al, 1981, J.biol. Chem. 256:3112), the Thermus thermophilus (Thermus thermophilus) (Tth) DNA polymerase (Myers and Gelfand 1991,Biochemistry 30:7661), the Bacillus stearothermophilus (Bacillus stearothermophilus) DNA polymerase (Stenesh and McGowan,1977,Biochim Biophys Acta 475:32), the Thermococcus thermophilus (Thermococcus litoralis) (Tli) DNA polymerase (also known as Vent DNA polymerase, cariello et al, 1991,Nucleic Acids Res,19:4193), thermotoga maritima (Thermotoga maritima) (Tma) DNA polymerase (Diaz and Sabino,1998Braz J.Med.Res,31:1239), thermus aquaticus (Taq) DNA polymerase (Chien et al, 1976, J. Bactoriol, 127:1550), thermococcus/Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et al, 1997, appl. Environ. Microbiol. 63:4504), JDF-3DNA polymerase (patent application WO 0132887) and Pyrococcus GB-D (PGB-D) DNA polymerase (Juncosa-Ginesta et al, 1994, biotech, 16:820). The polymerase activity of any of the above enzymes can be determined by methods well known in the art.

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. In general, a primer is specific for a target sequence if the primer-target duplex stability is greater than the stability of the duplex formed between the primer and any other sequences found in the sample. Those skilled in the art will recognize that various factors, such as salt conditions and base composition of the primer and location of mismatches, will affect the specificity of the primer and in many cases require routine experimental confirmation of primer specificity. Hybridization conditions may be selected under which the primer may form a stable duplex with only the target sequence. Thus, the use of target-specific primers under suitably stringent amplification conditions enables selective amplification of those target sequences that comprise the binding site for the target primer.

As used herein, the term "non-specific amplification" refers to the amplification of a nucleic acid sequence other than a target sequence that results from hybridization of a primer to the sequence other than the target sequence and then serves as a substrate for primer extension. Hybridization of a primer to a non-target sequence is referred to as "non-specific hybridization", particularly at lower temperatures, reduced stringency, pre-amplification conditions, or in the presence of variant alleles in a sample that have sequences very close to the true target, such as Single Nucleotide Polymorphisms (SNPs).

The term "3 '-mismatch discrimination" refers to a property of DNA polymerase that distinguishes a perfectly complementary sequence from a sequence that contains mismatches (nearly complementary), where the nucleic acid to be extended (e.g., primer or other oligonucleotide) has a mismatch at the 3' end of the nucleic acid as compared to the template to which the nucleic acid hybridizes. In some embodiments, the nucleic acid to be extended comprises a mismatch at the 3' end relative to the fully complementary sequence.

The term "3 '-mismatch discrimination assay" refers to an assay that discriminates for the presence of increased specificity in amplification of a target DNA sequence, except for the presence of one or more nucleotide residues having different base compositions at or near their respective 3' -ends, when the target DNA sequence is analyzed with two primers having substantially the same sequence. For example, a first primer having a 3 'terminal sequence that is fully complementary to a target DNA sequence is considered a 3' matched primer, while a second primer having a 3 'terminal sequence with at least one nucleotide base that is not complementary to the target DNA sequence is considered a 3' mismatched primer. Examples of 3' mismatch discrimination assays are provided in many examples (e.g., example 4 tables 10 and 11, etc.).

As used herein, the term "primer dimer" refers to a template-independent, non-specific amplification product that is believed to result from primer extension, wherein another primer is used as a template. Although primer dimers often appear as concatemers of two primers, i.e., dimers, concatemers of more than two primers also exist. The term "primer dimer" is generally used herein to encompass template-independent, non-specific amplification products.

As used herein, the term "reaction mixture" refers to a solution comprising reagents necessary to carry out a given reaction. "amplification reaction mixture" refers to a solution containing the reagents necessary to carry out the amplification reaction, typically containing oligonucleotide primers and DNA polymerase or ligase in a suitable buffer. The "PCR reaction mixture" typically comprises oligonucleotide primers, DNA polymerase (most typically thermostable DNA polymerase), dNTPs and divalent metal cations in a suitable buffer. If the reaction mixture contains all the reagents necessary to be able to carry out the reaction, it is referred to as a complete reaction mixture, and if only a subset of the necessary reagents is contained, it is referred to as an incomplete reaction mixture. It will be appreciated by those skilled in the art that for reasons of convenience, storage stability, or to allow adjustment of the concentration of the components depending on the application, the reaction components are typically stored as separate solutions, each solution containing a subset of the total components, and the reaction components are combined prior to reaction to produce the complete reaction mixture. Furthermore, one skilled in the art will appreciate that the reaction components are packaged separately for commercialization, and that a useful commercial kit may comprise any subset of reaction components including the blocked primers of the present invention.

For the purposes of the present invention, the term "non-activated" or "inactivated" as used herein refers to the inability of a primer or other oligonucleotide to participate in a primer extension reaction or ligation reaction because a DNA polymerase or DNA ligase is unable to interact with an oligonucleotide for its intended purpose. In some embodiments, when the oligonucleotide is a primer, the non-activated state occurs because the primer is blocked at or near the 3' end to prevent primer extension. When a specific group is bound at or near the 3' end of the primer, the DNA polymerase cannot bind to the primer and no extension occurs. However, the non-activating primer is capable of hybridizing to a substantially complementary nucleotide sequence.

For the purposes of the present invention, the term "activated" as used herein refers to a primer or other oligonucleotide capable of participating in a reaction with a DNA polymerase or DNA ligase. The primer or other oligonucleotide is activated upon hybridization to a substantially complementary nucleic acid sequence and cleaved to yield a functional 3 'end or 5' end, which can interact with a DNA polymerase or DNA ligase. For example, when the oligonucleotide is a primer and the primer hybridizes to the template, the 3 'blocking group may be removed from the primer by, for example, a cleaving enzyme, so that the DNA polymerase may bind to the 3' end of the primer and facilitate primer extension.

As used herein, the term "cleavage domain" or "cleavable domain" is synonymous and refers to a region located between the 5 'and 3' ends of a primer or other oligonucleotide that is recognized by a cleavage compound, such as a cleavage enzyme (which will cleave the primer or other oligonucleotide). For the purposes of the present invention, the cleavage domain is designed such that a primer or other oligonucleotide is cleaved only when it hybridizes to a complementary nucleic acid sequence, but is not cleaved when it is single stranded. The sequence of the cleavage domain or both sides thereof may comprise the following portions: a) preventing or inhibiting extension or ligation of primers or other oligonucleotides by a polymerase or ligase, b) enhancing discrimination to detect variant alleles, or c) inhibiting undesired cleavage reactions. One or more such portions may be included in the sequence of the cleavage domain or its flanking.

As used herein, the term "rnase H cleavage domain" is a cleavage domain comprising one or more ribonucleic acid residues or alternative analogues that provide substrates for rnase H. The RNase H cleavage domain may be located anywhere within the primer or oligonucleotide, preferably at or near the 3 'or 5' end of the molecule.

The "rnase H2 cleavage domain" may comprise one RNA residue, a sequence of consecutively 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' -fluoronucleoside residue. In a more preferred embodiment, the rnase H2 cleavable domain comprises two adjacent 2' -fluoro residues.

The term "blocked primer" as used herein refers to a primer having at least a cleavage domain suitable for sufficient hybridization to 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 propylene glycol (C3) spacer.

As used herein, the term "cleavage compound" or "cleavage agent" refers to any compound that can recognize a cleavage domain within a primer or other oligonucleotide and selectively cleave the oligonucleotide depending on the presence of the cleavage domain. Cleavage compounds used in the present invention selectively cleave primers or other oligonucleotides that contain a cleavage domain only upon hybridization to a substantially complementary nucleic acid sequence, but do not cleave primers or other oligonucleotides when the primers or other oligonucleotides are single stranded. The cleavage compound cleaves the primer or other oligonucleotide within or near the cleavage domain. The term "adjacent", as used herein, refers to cleavage of a primer or other oligonucleotide by a cleavage compound at the 5 'end or 3' end of the cleavage domain. The preferred cleavage reaction of the present invention produces a 5 '-phosphate group and a 3' -OH group.

In a preferred embodiment, the cleavage compound is a "cleavage enzyme". A cleaving enzyme is a protein or ribozyme that is capable of recognizing a cleavage domain when a primer or other nucleotide hybridizes to a substantially complementary nucleic acid sequence, but does not cleave the complementary nucleic acid sequence (i.e., it provides a single strand break in a duplex). When the primer or other oligonucleotide is single stranded, the cleavage enzyme will not cleave the primer or other oligonucleotide comprising the cleavage domain. Examples of cleavage enzymes are RNase H enzyme and other nicking enzymes.

As used herein, the term "nick" refers to the cleavage of only one strand of a double-stranded portion of a fully or partially double-stranded nucleic acid. The location at which the nucleic acid is nicked is referred to as the "nicking site" (NS). A "nicking agent" (NA) is a reagent that nicks a partially or fully double-stranded nucleic acid. It may be an enzyme or any other compound or composition. In certain embodiments, the nicking agent can recognize a particular nucleotide sequence of a fully or partially double-stranded nucleic acid and cleave only one strand of the fully or partially double-stranded nucleic acid at a particular 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).

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

As used herein, the term "blocking group" refers to a chemical moiety that binds to a primer or other oligonucleotide so that no amplification reaction occurs. For example, primer extension and/or DNA ligation does not occur. Once the blocking groups are removed from the primer or other oligonucleotide, the oligonucleotide can be involved in the assay (PCR, ligation, sequencing, etc.) of its design. Thus, a "blocking group" may be any chemical moiety that inhibits recognition by a polymerase or DNA ligase. Blocking groups may be incorporated into the cleavage domain, but are typically located either 5 'or 3' to the cleavage domain. The blocking group may be composed of more than one chemical moiety. In the present invention, the "blocking group" is typically removed after hybridization of the oligonucleotide to its target sequence.

The term "fluorogenic probe" refers to either a) an oligonucleotide having a linked fluorophore and quencher, and optionally a minor groove binder, or b) a DNA binding reagent, e.g Green dye.

The term "fluorescent label" or "fluorophore" refers to a compound having a maximum fluorescence emission between about 350 and 900 nm. A variety of fluorophores may be used, including but not limited to: 5-FAM (also known as 5-carboxyfluorescein; also known as spiro (isobenzofuran-1 (3H), 9' - (9H) xanthene) -5-carboxylic acid, 3',6' -dihydroxy-3-oxo-6-carboxyfluorescein); 5-hexachloro-fluorescein; ([ 4,7,2',4',5',7' -hexachloro- (3 ',6' -dipivaloyl-fluorescein yl) -6-carboxylic acid]) The method comprises the steps of carrying out a first treatment on the surface of the 6-hexachloro-fluorescein; ([ 4,7,2',4',5',7' -hexachloro- (3 ',6' -dipivaloyl) fluorescein) -5-carboxylic acid]) The method comprises the steps of carrying out a first treatment on the surface of the 5-tetrachlorofluorescein; ([ 4,7,2',7' -Tetrachloro- (3 ',6' -Dipivaloyl fluorescent radical) -5-formic acid]) The method comprises the steps of carrying out a first treatment on the surface of the 6-tetrachlorofluorescein; ([ 4,7,2',7' -Tetrachloro- (3 ',6' -Dipivaloyl luciferase) -6-carboxylic acid]) The method comprises the steps of carrying out a first treatment on the surface of the 5-TAMRA (5-carboxytetramethyl rhodamine); xanthium, 9- (2, 4-dicarboxyphenyl) -3, 6-bis (dimethylamino); 6-TAMRA (6-carboxytetramethyl rhodamine); 9- (2, 5-dicarboxyphenyl) -3, 6-bis (dimethylamino); EDANS (5- ((2-aminoethyl) amino) naphthalene-1-sulfonic acid); 1,5-IAEDANS (5- ((((2-iodoacetyl) amino) ethyl) amino) naphthalene-1-sulfonic acid); cy5 (indodicarboncyanine-5); cy3 (indodicarboncyanine-3); and BODIPY FL (2, 6-dibromo-4, 4-difluoro-5, 7-dimethyl-4-bora-3 a,4 a-diaza-s-indan-3-propionic acid); -670 dye (Biosearch Technologies); cal->Orange dye (Biosearch Technologies); a Rox dye; max dyes (Integrated DNA Technologies), and suitable derivatives thereof.

As used herein, the term "quencher" refers to a portion of a molecule or compound that is capable of reducing emission from a fluorescent donor when attached to or in proximity to the donor. Quenching may occur by any of several mechanisms, including fluorescence resonance energy transfer, photoinduced electron transfer, paramagnetic enhancement of intersystem crossing, and,Texel exchange coupling and exciton coupling, such as the 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 fluorescence in the absence of the quencher. Many commercially available quenchers are known in the art, including but not limited to DABCYL, blackHoleTM quenchers (BHQ-1, BHQ-2 and BHQ-3), iowaFQ and Iowa->RQ. These are so-called dark quenchers. They do not fluoresce naturally, almost eliminating the background problems seen with other quenchers (e.g., TAMRA, which themselves fluoresce).

As used herein, the term "ligation" refers to covalent ligation of two polynucleotide ends. In various embodiments, ligation involves covalently ligating the 3 'end of a first polynucleotide (acceptor) to the 5' end of a second polynucleotide (donor). Ligation results in the formation of phosphodiester bonds between the ends of the polynucleotides. In various embodiments, ligation may be mediated by any enzyme, chemical or process that results in covalent ligation of polynucleotide ends. In certain embodiments, the ligation is mediated by a ligase.

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 the ligase include E.coli DNA ligase, T4 DNA ligase and the like.

Ligation reactions can be used in DNA amplification methods, such as the "ligase chain reaction" (LCR), also known as the "ligase amplification reaction" (LAR), see Barany, proc. And Wu and Wallace, genomics 4:560 (1989), incorporated herein by reference. In LCR, four oligonucleotides, two adjacent oligonucleotides that uniquely hybridize to one strand of the target DNA, and a set of complementary adjacent oligonucleotides that hybridize to the opposite strand are mixed, and a DNA ligase is added to the mixture. In the presence of the target sequence, the DNA ligase will covalently link each set of hybrid molecules. Importantly, in LCR, two oligonucleotides will only ligate together when they are base-paired with the sequence without gaps. Repeated cycles of denaturation, hybridization and ligation can amplify a small piece of DNA. Mismatches at the junction between adjacent oligonucleotides inhibit ligation. This property allows the use of LCR to distinguish variant alleles, such as SNPs, as in other oligonucleotide ligation assays. LCR has also been used in conjunction with PCR to achieve enhanced detection of single base changes, see Segev, PCT publication No. WO9001069 (1990).

When the term "codon optimized" modifies a particular nucleic acid encoding a polypeptide, the term is meant to encompass preferred codons that are efficiently expressed in a given host cell, e.g., a given microorganism (e.g., E.coli, saccharomyces cerevisiae, etc.) or a mammalian cell (e.g., a human cell, e.g., heLa, COS cells, etc.). Such preferred codons are well known in the art based on codon bias tables developed for a variety of organisms. Polynucleotides encoding polypeptides of the invention include those having an open reading frame optimized for codons of any known organism in which codon-biased tables have been developed or for which such tables can be readily discerned from empirical assays.

The phrase "BaseX PCR amplification method" refers to a highly efficient nucleic acid amplification method that allows for more than 2-fold increase in amplification product per amplification cycle, and thus has higher sensitivity and speed than conventional PCR. This method is disclosed in U.S. patent publication US10273534 (B2) entitled "Exponential base-groter-than-2 nucleic acid amplification" to R.Higuchi (applicant: cepheid) at month 4 of 2019, the contents of which are incorporated herein by reference in their entirety.

The phrase "fusion protein" or "fusion polypeptide" refers to a protein comprising additional amino acid information that is not native to the protein to which the additional amino acid information is covalently linked. Such additional amino acid information may include tags that enable purification or identification of the fusion protein. Such additional amino acid information may include peptides that enable the fusion protein to be transported into the cell and/or to a specific location within the cell. Examples of labels for these purposes include: aviTag, a peptide that allows biotinylation of the BirA enzyme, so that the protein can be isolated by streptavidin; a Calmodulin-tag, which is a peptide bound by the protein Calmodulin; polyglutamic acid tags, which are peptides that bind efficiently to anion exchange resins (e.g., mono-Q); an E-tag, which is a peptide recognized by an antibody; a FLAG-tag, which is a peptide recognized by an antibody; an HA-tag, a peptide from hemagglutinin, which is recognized by an antibody; his tags, which are typically 5-10 histidines bound by nickel or cobalt chelates; myc-tag, a peptide derived from c-Myc, which is recognized by an antibody; NE-tag, a novel 18 amino acid synthetic peptide, which is recognized by monoclonal IgG1 antibodies, can be used in a wide variety of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins; an S-tag which is a peptide derived from RNase A; an SBP-tag, which is a peptide that binds to streptavidin; softag 1 for mammalian expression; softag 3 for prokaryotic expression; strep-tag, a peptide that binds to streptavidin or modified streptavidin called streptavidin (Strep-tag II); a TC tag, a tetracysteine tag, which is recognized by both FlAsH and ReAsH biarsen compounds; v5 tag, which is a peptide recognized by an antibody; a VSV-tag, which is a peptide recognized by an antibody; xpress tag; isopeptag, a peptide that is covalently bound to the pilin-C protein; spyTag, a peptide that covalently binds to SpyCatcher protein; snootag, a peptide covalently bound to the snootche protein; BCCP (biotin carboxyl carrier protein), a protein domain biotinylated by BirA, capable of recognition by streptavidin; a glutathione-S-transferase tag, which is a protein that binds to immobilized glutathione; a green fluorescent protein tag, which is an autofluorescent protein, that can be bound by an antibody; haloTag, a mutant bacterial haloalkane dehalogenase, which is covalently attached to reactive haloalkane substrates, thereby allowing attachment to a variety of substrates; a maltose binding protein tag, which is a protein that binds to amylose agarose; nus-tag; thioredoxin tags; and Fc-tags derived from immunoglobulin Fc domains, allowing dimerization and solubilization, useful for purification on protein-a sepharose. Nuclear Localization Signals (NLS), such as those obtained from SV40, allow proteins to be transported to the nucleus immediately after entering the cell. Given that the native Cas9 protein originates from bacteria, it does not naturally contain NLS motifs, the addition of one or more NLS motifs to the recombinant Cas9 protein is expected to exhibit improved genome editing activity when used with target genomic DNA substrates located in eukaryotic cells in the nucleus. Those of skill in the art will understand these different fusion tag techniques, the particular amino acid sequences involved, and how to make and use fusion proteins comprising them. In one embodiment, a highly preferred fusion protein or fusion polypeptide comprises a His-tag motif, although one skilled in the art will appreciate that other tags may be included, as described above. The invention includes fusion proteins or fusion polypeptides, and original mutated forms of the corresponding proteins or polypeptides lacking additional amino acid sequence information.

Novel hybrid rnase H2 enzyme variants produced by recombinant shuffling of amino acid sequences from known rnase H2 enzymes.

Two hybrid RNase H2 enzymes with novel useful properties were generated by partial recombination ("shuffling") of the amino acid sequences of three RNase H2 enzymes, including Thermococcus deep sea, thermococcus costa and Pyrococcus furiosus. And can produce hybrid RNase H2 enzyme. In particular, two mutant RNase H2 enzymes, SEL28 RNase H2 (SEQ ID NO: 89) and SEL29 RNase H2 (SEQ ID NO: 90), have been found to significantly enhance mismatch discrimination, combining amino acid sequence fragments from Thermococcus abyssal, thermococcus ida and Pyrococcus furiosus organisms. The mutants were selected from libraries created by randomly shuffling the rnase H2 sequences. Both mutant enzymes contained fragments of amino acid residues 26-40 and residues 100-120 of T.kod RNase H2, but other variations were also present. Based on structures with known crystal structuresHomology (Muroya et al, 2001; rychlik et al, 2010), these residues may be in contact with the bound DNA duplex. Without being bound by any particular theory, it is hypothesized that residues 26-40 and residues 100-120 of t.kod rnase H2 will alter the enzyme binding pocket of the substrate duplex, resulting in a change in binding affinity and catalyzing nucleic acid cleavage. 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 described in Table 1. Corresponding transgenes (His) of wild-type P.ab.RNase H2 protein (SEQ ID NO.: 1), hybrid SEL28 RNase H2 protein (SEQ ID NO.: 2) and hybrid SEL29 RNase H2 protein (SEQ ID NO.: 3) were also prepared ₆ The amino acid sequence of the tag, and serves as the basis for the production of additional mutant rnase H2 protein (see table 3).

TABLE 1 amino acid sequence of hybrid RNase H2 protein.

¹ The same amino acids as wild-type Thermococcus abyssal RNase H2 are shown as underlined, not bolded sequences. Amino acids derived from the sequences of Thermococcus costa and Pyrococcus furiosus RNase H2 are shown in bold and underlined, respectively.

The resulting hybrid SEL28 and SEL29 rnase H2 enzymes encoded by SEQ ID nos.: 89 and 90 improve mismatch discrimination when the mismatch is at an RNA nucleotide, but to a different extent and with different specificity. (data not shown). Likewise, the coding of the SEL28 and SEL29 mutant RNase H2 enzymes by SEQ ID NO. 89 and 90 improves the mismatch discrimination 5' of the RNA nucleotides. (data not shown.)

rhPCR can also be performed using high fidelity DNA polymerase-e.g., DNA polymerase from Pyrococcus furiosus and Thermococcus KOD-rather than DNA polymerase from Thermus aquaticus (Taq). However, WT p.a. rnase H2, SEL28 rnase H2 and SEL29 rnase H2 have limited activity in rhPCR using high-fidelity polymerase and related reaction buffers. The optimal reaction buffer for Taq DNA polymerase is very different from that for KOD DNA polymerase. It was found that the mutation in RNase H2 was more tolerant to the components in the KOD DNA polymerase reaction buffer. We show that the use of KOD DNA polymerase reaction buffer, Q48R, A V and P13S/A107V, enhances enzyme activity when added to the hybridization mutants SEL28 or SEL29 RNase H2.

The present invention relates to mutant rnase H2 enzymes that enhance enzyme activity during rhPCR using KOD DNA polymerase and its optimal reaction buffer. The 48 th and 107 th amino acid mutations of SEL29 rnase H2 have been shown to have this improved activity. Seven point mutants with background of SEL28 rnase H2 or SEL29 rnase H2 were screened and shown to enhance enzyme activity with Q48R SEL29 rnase H2 and a107V SEL29 rnase H2 using KOD DNA polymerase reaction buffer. These have also been shown to retain or enhance the mismatch discrimination ability of SEL29 rnase H2.

RNase H2-mediated PCR

The hybrid rnase H2 muteins 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 deep sea or related organisms and DNA primers comprising a single ribonucleotide residue and a 3' blocking moiety ("blocked cleavable primers"). The blocked cleavable primer is activated when cleaved by the rnase H2 enzyme. After hybridization of the primer to the target DNA, cleavage occurs 5' to the RNA base. Primer dimer is reduced because primers can only be cleaved after hybridization to a perfectly matched target sequence. The requirement for high target complementarity reduces the amplification of closely related sequences.

In this regard, the hybrid rnase H2 muteins are particularly suitable for enhancing the performance of generating high quality genomic amplicon libraries for high throughput multiplex sequencing applications, such as next generation sequencing applications (NGS). In particular, Q48R SEL29 is a useful RNase H2 enzyme for RNase H2-mediated PCR applications and systems, such as applicants' rhAmpSeq ^TM The system.

RNase H2-mediated SNP detection and rare allele detection

Because of its enhanced 3' mismatch discrimination properties, the hybrid rnase H2 muteins disclosed herein can be used for detecting single nucleotide polymorphism detection and rare allele detection. The use of blocked cleavable primers that form a complete duplex with only the desired SNP-containing nucleic acid template will be recognized and cleaved by the hybrid RNase H2 mutein, thereby activating the primer: the desired nucleic acid template duplex for primer extension by a DNA polymerase under appropriate conditions.

RNase H2 in loop-mediated isothermal amplification (LAMP)

The use of rnase H2 in loop-mediated isothermal amplification (LAMP) is also contemplated herein. The LAMP amplification method is performed under isothermal conditions, i.e., no change in reaction temperature occurs during cycling. LAMP requires the design of at least four different primers to recognize six different regions of the desired amplicon (Notomi et al Nucleic Acids Research,28 (12) (2000)). The amplification reaction depends on the strand displacement activity of a DNA polymerase usually derived from bacillus stearothermophilus (Bst). The structure of the product consists of inverted repeat long chains of the target sequence.

Because of the large number of primers and the use of mesophilic DNA polymerase in this method, the LAMP reaction is prone to form primer dimer products. LAMP also lacks 5'- >3' exonuclease activity in amplifying BST polymerase, as this activity can disrupt amplification by competing with the necessary strand displacement activity. The use of blocked cleavable primers and rnase H2 can reduce or eliminate detection of primer dimer signals in LAMP reactions. In this regard, the hybrid rnase H2 mutein is particularly suitable for enhancing the performance of the desired product formed in the LAMP reaction without concomitant production of primer dimer.

Application of

In a first aspect, a hybrid rnase H2 protein is provided. Hybrid rnase H2 proteins include amino acid sequence fragments from the organisms pyrococcus abyssal (Pyrococcus abyssi) (p.a.), pyrococcus costa (t.kod) and pyrococcus intensiviss (Pyrococcus furiosus). In a first aspect, the hybrid RNase H2 protein comprises amino acid residues 26-40 and residues 100-120 of T.kod RNase H2. In a second aspect, the hybrid RNase H2 protein is selected from SEQ ID NOs 2 and 3. In a third aspect, the hybrid RNase H2 protein is selected from SEQ ID NOS.14-20.

In a second aspect, there is provided a recombinant nucleic acid encoding any of the hybrid rnase H2 proteins disclosed herein. In a first aspect, exemplary recombinant nucleic acids encoding any hybrid RNase H2 protein include SEQ ID NOs 79-87 of Table 14.

In a third aspect, a method of performing primer extension is provided. The method comprises the following steps: contacting a hybrid rnase H2 protein disclosed herein with a primer, a polynucleotide template, nucleoside triphosphates, and a DNA polymerase under conditions suitable for a primer extension method, thereby producing an extended primer. In a first aspect, the DNA polymerase comprises a high fidelity archaea DNA polymerase. In a second aspect, the primer comprises a blocked cleavable primer. In a third aspect, the primer extension method comprises a method of performing a Polymerase Chain Reaction (PCR). In a fourth aspect, the method of performing PCR improves mismatch discrimination in a primer:polynucleotide hybrid formed between the primer and the polynucleotide template. In a fifth aspect, the improvement in mismatch discrimination comprises an improvement in 3' -mismatch discrimination.

In a fourth aspect, a reaction mixture is provided. The reaction mixture includes the hybrid rnase H2 protein described herein, at least one primer, a polynucleotide template, nucleoside triphosphates, and a DNA polymerase. In a first aspect, the reaction mixture includes a DNA polymerase that is a high fidelity archaea DNA polymerase. In a second aspect, the reaction mixture includes at least one primer that is a blocked cleavable primer.

In a fifth aspect, a method for performing rhPCR is provided. The method comprises the step of primer extension with the hybrid rnase H2, DNA polymerase and primers described herein. In a first aspect, a method of performing rhPCR comprises primer extension with a high fidelity archaea DNA polymerase. In a second aspect, the hybrid rnase H2 enzyme is reversibly inactivated by chemical modification, aptamer or blocking antibody. In a third aspect, the blocking group is attached to the 3' terminal nucleotide of the primer. In a fourth aspect, a blocking group is attached to the 5 'end of the 3' terminal residue and inhibits the primer from acting as a template for DNA synthesis. In a fifth aspect, the blocking group comprises one or more abasic residues. In a sixth aspect, one or more abasic residues is a C3 spacer. In a seventh aspect, 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 degradation resistant proprietary group. In this regard, blocking groups include labels that allow detection of extension amplification reactions. In this regard, a label allowing detection of the amplification reaction is attached to the oligonucleotide primer 3' of the cleavage site. In this regard, the label is a fluorophore or a mass label for mass spectrometric detection of the amplification reaction. In a further aspect, the cleavage domain of the blocked cleavable primer comprises one or more of the following: DNA residues, abasic residues, modified nucleosides or modified internucleotide linkages. In further aspects, the cleavage domain comprises a single RNA residue, two adjacent RNA residues, a contiguous sequence of three or more RNA residues, a lack of RNA residues, or one or more 2' -modified nucleosides. In those aspects, wherein the cleavage domain comprises one or more 2' -modified nucleosides 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' -fluoronucleosides.

In a sixth aspect, a method of amplifying a target DNA sequence is provided. The method comprises several steps. The first step is to provide a reaction mixture comprising: (i) An oligonucleotide primer having a cleavage domain cleavable by an rnase H2 enzyme, located 5 'of a blocking group attached at or near the 3' end of the oligonucleotide primer, wherein the blocking group prevents primer extension and/or inhibits oligonucleotide primer as a template for DNA synthesis; (ii) a sample nucleic acid which may or may not be a target sequence; (iii) A DNA polymerase, and (iv) a hybrid rnase H2 protein disclosed herein. The second step is to hybridize the oligonucleotide primer to 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 aspect of the method, the DNA polymerase is an archaea high-fidelity DNA polymerase. On the other hand, RNase H2 protein is reversibly inactivated by chemical modification or blocking antibodies. In a further aspect of the method, the blocking group is attached to the 3' terminal nucleotide of the oligonucleotide primer. In other aspects of the method, a blocking group is attached to the 5 'end of the 3' terminal residue and inhibits the oligonucleotide primer from acting as a template for DNA synthesis. In a further aspect of the method, the blocking group comprises one or more abasic residues. In further aspects of the method, one or more abasic residues are a C3 spacer or other degradation resistant proprietary group. In other 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 degradation resistant proprietary group. In a further aspect of the method, the blocking group comprises a label that allows detection of an extension amplification reaction. In a further aspect of the method, the method further comprises a label allowing detection of the amplification reaction, wherein the label is attached to the oligonucleotide primer 3' to the cleavage site. In these aspects, the label is a fluorophore or a mass label for detection of the amplification reaction by mass spectrometry. In other aspects of the method, the cleavage domain comprises one or more of the following: DNA residues, abasic residues, modified nucleosides or modified internucleotide linkages. In further aspects of the method, the cleavage domain comprises a single RNA residue, two adjacent RNA residues, a contiguous sequence of three or more RNA residues, or one or more 2' -modified nucleosides. In terms of methods in which the cleavage domain comprises one or more 2 '-modified nucleosides, those 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' -fluoronucleosides.

In a seventh aspect, a kit for generating an extended primer is provided. The kit comprises at least one container that provides a 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) Providing a container of primers which hybridize to a predetermined polynucleotide template under primer extension conditions; (b) providing a container of nucleoside triphosphates; (c) Providing a container of buffer suitable for primer extension and (d) a DNA polymerase. In a second aspect, the DNA polymerase comprises a high fidelity archaea DNA polymerase. In a third aspect, the kit comprises one or more additional containers containing blocked cleavable primers.

In an eighth aspect, a kit for performing amplification of a target DNA sequence is provided. The kit includes a reaction buffer comprising rnase H2 and a high fidelity archaea DNA polymerase as described herein. In a first aspect, the kit further comprises one or more oligonucleotide primers, wherein at least one oligonucleotide primer has a cleavage domain cleavable by an rnase H2 enzyme, located 5 'of a blocking group attached at or near the 3' end of the oligonucleotide primer, wherein the blocking group prevents primer extension and/or inhibits oligonucleotide primer from acting as a template for DNA synthesis. In a second aspect, the kit comprises a blocking group that 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 degradation resistant proprietary group.

In a ninth aspect, a method of preparing a library of template nucleic acid amplicons is provided. The method comprises several steps. The first step is to form a mixture comprising a population of nucleic acids, at least a blocked cleavable primer, a hybrid rnase H2 protein, dntps, a DNA polymerase, and a buffer, such that hybridization duplex is formed between at least the blocked cleavable primer and the population of nucleic acids in the mixture. The second step is cleaving the at least one blocked cleavable primer with a hybrid rnase H2 protein to produce 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 in the buffer with the DNA polymerase under conditions that allow for amplification of one or more template nucleic acids from the population of nucleic acids, thereby generating amplicons of the template nucleic acids. In a first aspect, the hybrid RNase H2 protein is selected from Q48R SEL29 (SEQ ID NO: 18) or others. In a second aspect, the DNA polymerase is a high fidelity archaea DNA polymerase or other. In a third aspect, the buffer is a high fidelity archaea DNA polymerase buffer.

In a thirteenth aspect, a method of rhPCR assay with reduced primer dimer formation is provided. The method comprises primer extension with Q48R SEL29 RNase H2 (SEQ ID NO: 18). The reduced primer dimer formation corresponds to the amount of primer dimer that was reduced during the rhPCR assay with Q48R SEL29 RNase H2 (SEQ ID NO: 18) when compared to the rhPCR assay with wild-type P.a.RNase H2 (SEQ ID NO: 1).

In a fourteenth aspect, a method of rhPCR assay is provided having improved mapping and targeting rates for desired products. The method comprises primer extension with Q48R SEL29 RNase H2 (SEQ ID NO: 18). The improved mapping and targeting rates correspond to increased mapping and targeting amplification of the desired product formed during the rhPCR assay with Q48R SEL29 rnase H2 (SEQ ID NO: 18) when compared to the rhPCR assay with wild-type p.a. rnase H2 (SEQ ID NO: 1).

Finally, the rnase H2 polypeptides of the invention are suitable for use in a BaseX PCR amplification method, which is a highly efficient amplification method disclosed in U.S. patent publication No. 10273534 (B2), the contents of which are incorporated herein by reference in their entirety.

Examples

The invention is further illustrated with reference to the following examples. It should be noted, however, that these examples, as with the embodiments described above, are illustrative and should not be construed as limiting the practicable scope of the invention in any way.

Example 1. Production of SEL28 and SEL29 hybrid rnase H2 proteins by recombinant shuffling.

The mutant RNase H2 protein was synthesized using in vitro DNA recombination and directed molecular evolution techniques. Altravax ^TM Inc (Sunnyvale, CA) generated a library containing 5,500 mutants according to the contractual agreement with Integrated DNA Technologies. SEL28 and SEL29 mutants were selected in a preliminary selection performed in IDT, where the mutants showed increased mismatch discrimination in the rnase H2 cleavage reaction. Mutants were generated in pET-27b (+) plasmid vectors in E.coli (E.coli) BL21 (DE 3). Proteins expressed based on the T7 system contain an N-terminal pelB signal sequence, a mutated RNase H2 gene, a human herpes simplex virus 2 epitope tag and a C-terminal hexahistidine tag. Using 50mL of TPP Bioreactor (Techno Plastic Products AG, trasadingen, switzerland) in a solution containing 50. Mu.g/mL kanamycin (Teknova) ^TM Coli cells were grown in 12mL of LB as well medium from Hollister, CA). Using Overnight Express ^TM Self-induction System 1 (MilliporeSigma) ^TM Burlington, MA) induced the expression of rnase H2 for 20 hours at 37 ℃. Using MaxQ ^TM 4000 track table (ThermoFisher Scientific) ^TM Grand Island, NY) were grown with shaking at 250 rpm. Cell at ThermoScientific Sorvall ^TM Centrifuge in a Legend XTR centrifuge at 7,500Xg for 10 minutes, discard supernatant. The cell paste was stored at-80℃and resuspended in 0.6mL of lysis buffer consisting of 50mM NaCl, 40mM Tris-HCl pH 8.0, 2.5mM MgCl2, 0.5mM CaCl2, 1 x->Extraction reagent (Millipore Sigma) ^TM ，Burlington，MA)、1x cOmplete ^TM EDTA-free protease inhibitor cocktail (Millipore Sigma, burlington, mass.), 0.1mg/mL lysozyme (. About.600 units, thermoFisher) ^TM Scientific, grand Island, N.Y.) and 4U/mL Ambion ^TM DNase I (ThermoFisher) ^TM Scientific, grand Island, N.Y.). Lysis at 25℃takes 15 minutes while shaking the cells at 120 rpm. Insoluble material was removed by centrifugation at 16,000Xg for 20 minutes. Dnase I and native escherichia coli protein were denatured at 75 ℃ for 15 min. Insoluble denatured proteins were removed again by centrifugation at 16,000Xg for 15 minutes. Capturem ^TM His-Tagged Miniprep kit (Takara Bio) ^TM Mountain View, CA) is used for protein purification. The binding column in the kit was washed with cell resuspension buffer and the supernatant containing rnase H2 was loaded onto the column. The solution was pulled through the column by centrifugation at 11,000Xg for 1 minute. The column was washed twice with 200. Mu.L of wash buffer (20 mM Na3PO4, 150mM NaCl, pH 7.6) plus 20mM imidazole. RNase H2 enzyme was eluted with 200. Mu.L of elution buffer (20 mM Na3PO4, 500mM NaCl, 500mM imidazole, pH 7.6), and 1L of 2 Xstorage buffer F (pH 8.4, 40mM Tris-HCl,0.2mM EDTA,200mM KCl) was used with D-Tube ^TM Dialyzer Midi, MWCO 6-8kDa (MilliporeSigma, burlington, mass.) was dialyzed overnight. The dialysis buffer in the tank is replaced at least once. Samples were recovered from the dialysis and mixed with a mixture of 99.8% (v/v) glycerol and 0.2% (v/v) Triton X-100 in a 1:1 volume ratio. These purified and concentrated RNase H2 solutions were stored at-20 ℃. Using Any kD ^TM Mini-/>TGX Stain-Free ^TM Protein gel (Bio- & gt>Hercules, CA), their purity was estimated by SDS gel electrophoresis. And Precision Plus Protein ^TM Undyed standard (Bio-Hercules, CA) shows dominant protein bands in all purified samples >75%) whose position corresponds to the expected molecular weight of 28.9 kg/mol.

The amino acid sequence of the mutein is shown in Table 1, SEQ ID NO:89 and 90.Sequencing was accomplished using the Applied Biosystems BigDye Terminators v 3.1.3.1 kit. According to the manufacturer's scheme, usePlus SV Minipreps DNA purification System (Promega, madison, wis.) plasmid DNA was isolated from bacterial strains. Sequencing data were collected by Applied Biosystems 3130 genetic analyzer.

Example 2 the Q48R and A107V SDM mutants in SEL29 RNase H2 increased the enzymatic activity in the DNA polymerase reaction buffer of Thermococcus costa compared to SEL29 RNase H2.

Generation of menses (His) by site-directed mutagenesis (SDM) techniques ₆ Labeled mutants SEL28 and SEL29 RNase H2 proteins (see, e.g., weiner M. Et al, gene,151:119-123 (1994)). Primers for SEL28 and SEL29 RNase H2 SDM are shown in Table 2, SEQ ID NOS: 4-13. The mutants were sequence verified and proteins were expressed in E.coli using standard methods. As previously described (Dobosy et al, 2011,and US patent 8,911,948B2), by charging Ni ²⁺ Affinity purification of the column the purification was completed. The amino acid sequences of the muteins are shown in Table 3, SEQ ID NOs 2-3, 14-20.

Table 2 SDM primers used for mutagenesis of RNase H2 enzyme.

¹ All bases are DNA. The characters shown in bold and underlined are mutagenic nucleotides.

Table 3. Amino acid sequence of RNase H2 protein.

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¹ The location of the mutation is shown in bold and underlined. Ending extension sum (His) ₆ The labels are shown in italics.

To determine if P13S, A V and P13S/A107V SEL28 RNase H2 or P13S, Q48R, A V or P13S/A107V SEL29 RNase H2 have increased activity in KOD DNA polymerase reaction buffers using rhPCR as compared to WT P.a RNase H2, quantitative rhPCR assays targeting the rs4939827SNP in SMAD7 were designed. This SNP has been used in the past (Dobosy et al, 2011 and U.S. Pat. No. 8,911,948B2) to characterize the efficiency and specificity of rhPCR and its reaction under different conditions is well known. The primers used in this assay are shown in Table 4, SEQ 21-23. The assay was performed at a reaction volume of 10 μl. Thermal cycle and data collectionReal-time system (Bio- & gt>Hercules, CA). Briefly, 200nM (2 pmol) of blocked forward primer (SEQ ID NO: 22) and 200nM (2 pmol) of unblocked reverse primer (SEQ ID NO: 23), or 200nM (2 pmol) of unblocked forward primer (SEQ ID NO: 21) and 200nM (2 pmol) of unblocked reverse primer (SEQ ID NO: 23) were combined with 2.5mM (total) MgSO ₄ 0.2mM (each) dNTP (MilliporeSigma) ^TM Burlington, mass.) and 0.5x +.>Dyes (Biotium inc., fremont, CA) were mixed into 1x internal KOD buffer (ROKStar buffer v2.0 or v 1.66) (IDT, coralville, IA). RNase H2 dilution buffer (IDT, coralville, IA) or 21fmol WT P.a. (SEQ ID NO: 1), SEL28 (SEQ ID NO: 2), SEL29 (SEQ ID NO: 3), P13S SEL28 (SEQ ID NO: 14), A107VSEL28 (SEQ ID NO: 15), P13S/A107V SEL28 (SEQ ID NO: 16), P13S SEL29 (SEQ ID NO: 17), Q48R SEL29 (SEQ ID NO: 18), A107V SEL29 (SEQ ID NO: 19) or P13S/A107V SEL29 (SEQ ID NO: 20) RNase H2 enzyme was added to each reaction. 10ng of genomic cell line DNA (cell line NA12878, coriell Institute for Medical Research, camden, NJ) representing the homozygous genotype for the rs4939827SNP was added to each reaction. Reactions were performed in triplicate and the results averaged. The reaction was cycled under the following conditions: 95 DEG C ^3:00 ->(95℃ ^0:10 ->60℃ ^0:30 ) x 75. Collecting embedded +.>Fluorescence data of (2). After the measurement is completed, the data are analyzed by Bio-Rad CFX +.>Automatic calling function calculation C of software _q Values. The results are presented in table 5.

Table 4. The sequences and SEQ ID of the primers used in the experiments described in example 2.

¹ DNA uppercase, RNA lowercase. X = a proprietary blocker group resistant to exonucleases.

TABLE 5 experiment C in example 2 _q 、ΔC _q And DeltaDeltaC _q Values.

Table 5 (subsequent)

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¹ ΔC _q The value was calculated as C of the blocked primer (SEQ ID NO: 22) _q Value and unblocked primer (SEQ ID NO: 21) C _q Differences between the values. ² ΔΔC _q The value was calculated as ΔC of the background SEL28 or SEL29 RNase H2 _q Delta C of the value and mutant SEL28 or SEL29 RNase H2 _q Differences between the values.

These data indicate that Q48R SEL29 and a107VSEL29 rnase H2 have increased enzymatic activity in the kodakohot coccus DNA polymerase reaction buffer compared to background SEL29 rnase H2, but P13S SEL29 rnase H2 and P13S/a107V rnase H2 are absent. Furthermore, P13S SEL28, A107V SEL28 and P13S/A107V RNase H2 did not increase enzyme activity in the DNA polymerase reaction buffer of Thermococcus costa compared to background SEL28 RNase H2. In the absence of any addition of RNase H2, the blocked primer is not cleaved and thus PCR cannot be supported. The use of proprietary anti-exonuclease blocking primers is necessary to prevent the primers from being blocked by high fidelity DNA polymerase. WT (p.a.) is able to cleave blocked primers compared to unblocked primers, but amplification is delayed, resulting in Δc _q 12.4 cycles in ROKstar buffer v2.0 and 21.1 cycles in ROKstar buffer v 1.66. Amplification delayed ΔC _q The quantification increased to 17.1 cycles of SEL28 rnase H2 and 24.6 cycles of SEL29 rnase H2 in ROKstar buffer v2.0, to 30.6 cycles of SEL28 rnase H2 and 30.3 cycles of SEL29 rnase H2 in ROKstar buffer v 1.66. Amplification delayed ΔC _q Quantification decreased with Q48R SEL29 RNase H2 (17.2 cycles in ROKstar buffer V2.0, 25.4 cycles in ROKstar buffer V1.66) and A107V RNase H2 (13.9 cycles in ROKstar buffer V2.0 and 21.9 cycles in ROKstar buffer V1.66). ΔC delayed in amplification by P13S/A107V SEL29 RNase H2 _q Quantification increased to 26.9 cycles in ROKstar buffer v2.0 and 32.8 in ROKstar buffer v1.66And each cycle. Neither P13S SEL28 nor P13S SEL29 RNase H2 had significant activity in ROKstar buffer v2.0 or v 1.66. Furthermore, P13S/A107VSEL28 RNase H2 delays amplification by ΔC _q Quantification increased to 21.5 cycles in ROKstar buffer v2.0 and 31.8 cycles in ROKstar buffer v 1.66. a107V SEL28 rnase H2 will amplify delayed Δc _q The quantification increased to 20.0 cycles in ROKstar buffer v2.0 and 35.8 cycles in ROKstar buffer v 1.66. These results are in contrast to the mutation of p.a.rnase H2 because Q48R, A V and P13S/a107V p.a.rnase H2 have increased enzymatic activity in the kodaka hot coccus DNA polymerase reaction buffer and P13S p.a.rnase H2 has lower but measurable enzymatic activity in the kodaka hot coccus DNA polymerase reaction buffer. Q48R SEL29 and A107V SEL29 RNase H2 increased enzyme activity-albeit to a different extent-when used with the Thermococcus costa DNA polymerase reaction buffer.

Example 3. Q48R SEL29 and A107V SEL29 RNase H2 increased mismatch discrimination when the mismatch was opposite the RNA position as compared to WT RNase H2.

The specific activity of the enzyme was determined using a fluorescence-based kinetic assay. The sequences of the DNA substrates are shown in Table 6, SEQ ID NOS.24-25. The substrate is a DNA hairpin with matching RNA bases in the double-stranded region. Attached to the 3' end of the probe is 6-FAM (6-carboxyfluorescein); attached to the 5' end of the probe is IowaFQ (SEQ ID NO.: 24). Fluorescence of 6-FAM is detected by Iowa +.in the complete hairpin probe>FQ moiety quenching. RNase H2 cleaves the 5 'end of the RNA base and releases the 3' end of the probe with 6-FAM. Thus, the fluorescence of 6-FAM is no longer quenched and can be emitted. DNA hairpins with RNA bases but without fluorophores or quenchers were used as competitors (SEQ ID NO.: 25). The assay was performed in a reaction volume of 10 μl. Use->480II (Roche Life Science, indianapolis, ind.) for data collection. Briefly, 1x rhAmp ^TM Backbone v3 was used in combination with 200nM (2 pmol) of the labeled hairpin (SEQ ID NO.: 24) and 10. Mu.M (100 pmol) of the competing hairpin (SEQ ID NO.: 25). 0.5, 1.0, 2.0 or 5.0fmol of WT P.a.RNase H2 (SEQ ID NO.: 1) or mutant RNase H2 (SEQ ID NO.: 18-19) was added to each reaction. The reaction was initially maintained at 4 ℃ to prevent cleavage of the substrate prior to starting the assay. The samples were run at 65 ℃. The fluorescence excitation wavelength is 483nm; the fluorescence emission wavelength was 533nm. Fluorescence intensity was collected every 13.75 seconds for 135 minutes. The initial velocity and the velocity per femtomole of each reaction were calculated. The per femtomole speed standard for each mutant was normalized to the value of WT P.a rnase H2 (whose specific activity was previously determined to be 17 units/μg enzyme). The specific activity of Q48R SEL29 RNase H2 was 46.03 units/. Mu.g of enzyme, and the specific activity of A107V SEL29 RNase H2 was 7.77 units/. Mu.g of enzyme.

Table 6. Sequences of DNA hairpins and SEQ ID for determining the activity of RNase H2 units.

¹ DNA uppercase, RNA lowercase. Q=iowaFQ. f=6-FAM (fluorescein).

To fully determine whether these mutated rnase H2 enzymes can improve mismatch discrimination of mismatches directly opposite RNA bases, synthetic quantitative rhPCR assays were used as described previously (Dobosy et al, 2011 and us patent 8,911,948B2). The assay can directly compare the effect of each specific single base mismatch as 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 at a reaction volume of 10 μl. Thermal cycle and data collectionReal-time system (Bio- & gt>Hercules, CA). Briefly, 200nM (2 pmol) of blocked reverse primer (SEQ ID NO: 28-31) and 200nM (2 pmol) of unblocked forward primer (SEQ ID NO: 26), or 200nM (2 pmol) of unblocked reverse primer (SEQ ID NO: 27) and 200nM (2 pmol) of unblocked forward primer (SEQ ID NO: 26) were mixed to 1x iQ ^TM />Green/>(Bio-/>Hercules, CA). To each reaction, 5mU of WT P.a. (SEQ ID NO: 1), 40mU of Q48R SEL29 (SEQ ID NO: 18) or 5mU of A107V SEL29 (SEQ ID NO: 19) RNase H2 enzyme was added. 20,000 copies of each synthetic target sequence (SEQ ID NOS: 32-35), each having a different nucleotide directly opposite the RNA base, were added to each reaction. Reactions were performed in triplicate and the results averaged. The reaction was cycled under the following conditions: 95 DEG C ^3:00 →(95℃ ^0:10 →60℃ ^0:30 ) x 75. Collecting embedded +.>Fluorescence data of Green. After the assay is complete, the data is analyzed and the average C for each paired combination is calculated _q And DeltaC _q Values. The results are provided in tables 8 and 9.

Table 7. Sequences of primers and templates used in the experiments described in example 2 and SEQ ID.

¹ DNA uppercase, RNA lowercase. X=c3 spacer (propylene glycol) blocking group. The positions of the mismatches are shown in bold and underlined in the synthetic template.

TABLE 8 experiment C described in example 2 _q Values.

Table 8 (subsequent)

TABLE 9 DeltaC of the experiment described in example 2 _q Values.

Watch 9 (subsequent)

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Watch 9 (subsequent)

ΔC from Table 9 _q The value was calculated as C for the nucleotide specific primer of each template _q C of value and perfect match template _q Differences between the values.

These data indicate that mismatch discrimination is greatly increased (about 14.0 cycles and about 13.5 cycles, respectively) when the mismatch is opposed to rC of Q48R SEL29 and A107V SEL29 RNase H2. In particular, there was a large increase in mismatch discrimination for the rC: C pair of Q48R SEL29 and A107V SEL29 RNase H2 (21.9 cycles and 15.2 cycles, respectively). Mismatch discrimination of Q48RSEL29 rnase H2 was significantly increased (about 11.2 cycles) when the mismatch was opposed to rU, but the increase in a107V SEL29 rnase H2 (about 4.3 cycles) was less significant. Mismatch discrimination of Q48R SEL29 and a107V SEL29 rnase H2 was significantly altered (about 8.5 cycles and about 6.0 cycles, respectively) when the mismatch was opposite to rA. Mismatch discrimination does not change much when mismatching is opposite to rG; this lack of variation in mismatch discrimination is less important, as mismatch discrimination is quite good for rG mismatches when using WT RNase H2. These increases in mismatch discrimination of Q48R SEL29 and a107V SEL29 rnase H2 are similar to those shown for background SEL29 rnase H2. When mismatching is directly opposite to the RNA bases, both mutations improve mismatch discrimination, but to different extents and specificities.

Example 4Q 48R SEL29 and A107V SEL29 RNase H2 increased mismatch discrimination when the mismatch was located 5' of the RNA compared to WT RNase H2.

To determine the degree of mismatch discrimination when the mismatch is located 5 'of the RNA nucleotide using Q48R SEL29 and a107V SEL29 rnase H2 enzyme, an assay was designed that targets rs113488022 (V600E SNP in the human BRAF gene), where the SNP is located 5' next to the RNA. The primers used in this assay are shown in Table 10, SEQ ID Nos. 36-39. Measurement of the reaction volume at 10. Mu.LAnd (3) row. Thermal cycle and data collectionReal-time system (Bio- & gt>Hercules, CA). Briefly, 200nM (2 pmol) of blocked forward primer (SEQ ID NO:38 or 39) and 200nM (2 pmol) of unblocked reverse primer (SEQ ID NO: 37), or 200nM (2 pmol) of unblocked forward primer (SEQ ID NO: 36) and 200nM (2 pmol) of unblocked reverse primer (SEQ ID NO: 37) were mixed to 1x iQ ^TM />Green/>Is a kind of medium. To each reaction, 5mU or 10mU of WT (SEQ ID NO.: 1) P.a.RNase H2 enzyme, 5mU or 10mU of SEL29 (SEQ ID NO.: 3) RNase H2 enzyme, 20mU or 40mU of Q48R SEL29 (SEQ ID NO.: 18) RNase H2 enzyme, or 5mU or 10mU of A107V SEL29 (SEQ ID NO.: 19) RNase H2 was added. Will synthesize double strand->2,000 copies of the (IDT, coralville, IA) template were added to each reaction, each corresponding to the homozygous genotype at the rs113488022SNP (SEQ ID NO:40 or 41). Reactions were performed in triplicate. The reaction was cycled under the following conditions: 95 DEG C ^3:00 →(95℃ ^0:10 →60℃ ^0:30 ) x 65. Collecting embedded +.>Fluorescence data of Green. After the assay is complete, the data is analyzed and the average C for each paired combination is calculated _q And DeltaC _q Values. The results are presented in table 11.

Table 10. Sequences of primers and templates used in the experiments described in example 4 and SEQ ID.

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¹ DNA uppercase, RNA lowercase. X=c3 spacer (propylene glycol) blocking group. The position of the mismatch is inAre shown in bold and underlined.

TABLE 11 experiment C in example 4 _q And DeltaC _q Values.

Table 11 (subsequent)

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These data indicate that Q48R SEL29 and a107V SEL29 rnase H2 significantly improved mismatch discrimination 5' of RNA nucleotides. ΔC of TrG primer _q Quantification increased from 3.8 cycles with WT P.a rnase H2 to 5.9 cycles with a107V SEL29 rnase H2 (for 5mU of WT rnase H2 and a107V SEL29 rnase H2) and from 4.5 cycles with wtp.a. rnase H2 to 6.9 cycles with Q48R SEL29 rnase H2 (for 5mU of WT rnase H2 and 40mU of Q48R SEL29 rnase H2). ΔC of ArG primer _q Quantification increased from 10.1 cycles with WT P.a RNase H2 to 11.2 cycles with A107V SEL29 RNase H2 (for 5mU of WT RNase H2 and A107V SEL29 RNase H2), but from when WTP.a.RNase H2 was used To 10.2 cycles when using Q48R SEL29 RNase H2 (for 5mU of WT RNase H2 and 40mU of Q48R SEL29 RNase H2). The variation in mismatch discrimination with ArG primer is less important because of the ΔC when using WT enzyme _q Has been quite effective for this primer. These increases in mismatch discrimination of Q48R SEL29 and a107V SEL29 rnase H2 are similar to those shown for background SEL29 rnase H2. Thus, Q48R SEL29 and a107VSEL29 rnase H2 can enhance mismatch discrimination when the mismatch is located 5' of the RNA base.

Example 5Q 48R SEL29 and A107V SEL29 RNase H2 increased mismatch discrimination when the mismatch was located 3' of the RNA compared to WT RNase H2.

To determine if the Q48R SEL29 and A107V SEL29 RNase H2 enzymes can improve mismatch discrimination when the mismatch is located 3 'of the RNA nucleotide, an assay was designed for the rs7583169 and rs3117947 SNPs, where the mismatch is located 3' of the RNA. The primers used in this assay are shown in Table 12, SEQ NO: 42-47. The assay was performed at a reaction volume of 10 μl. Thermal cycle and data collectionReal-time system (Bio- & gt>Hercules, CA). Briefly, 200nM (2 pmol) of blocked forward primer (SEQ ID NO:43 or 46) and 200nM (2 pmol) of unblocked reverse primer (SEQ ID NO:44 or 47), or 200nM (2 pmol) of unblocked forward primer (SEQ ID NOs.:42 or 45) and 200nM (2 pmol) of unblocked reverse primer (SEQ ID NOs.:44 or 47) were mixed to 1 X. >In a gene expression premix (IDT, coralville, IA) containing 3.0mM (total) MgCl ₂ And 0.5 x->Dyes (Biotium inc., fremont, CA). 5mU or 10mU of WT (SEQ ID NO.: 1) P.a.RNase H2 enzyme, 50mU or 100mU of Q48R SEL29 (SEQ ID NO.: 18)) RNase H2 enzyme or 20mU or 40mU A107V SEL29 (SEQ ID NO: 19) RNase H2 enzyme was added to each reaction. 20ng of genomic cell line DNA (cell lines NA12878 and NA24385, coriell Institute for Medical Research, camden, NJ) was added to each reaction, cell lines NA12878 and NA24385 representing the two homozygous genotypes at the rs7583169 and rs3117947 SNPs. Reactions were performed in triplicate and the results averaged. The reaction was cycled under the following conditions: 95 DEG C ^3:00 ->(95℃ ^0:10 ->60℃ ^0:30 ) x 65. Collecting embedded +.>Fluorescence data of (2). After the assay is complete, the data is analyzed and the average C for each paired combination is calculated _q And DeltaC _q Values. The results are presented in table 13.

Table 12. Sequences and SEQ ID of primers used in the experiments described in example 5.

TABLE 13 experiment C in example 5 _q And DeltaC _q Values.

ΔC from Table 13 _q The value was calculated as C of the mismatched template (NA 24385) _q C of value and match template (NA 12878) _q Differences between the values. Data for SEL29 rnase H2 were previously collected.

These data indicate that Q48R SEL29 and a107V SEL29 significantly increase mismatch discrimination 3' of RNA nucleotides. rs7583169 mismatch discrimination ΔC of SNP _q Quantification increased from 0.0 cycles with WT P.a rnase H2 to 1.4 cycles with a107V SEL29 rnase H2 (for 5mU of WT rnase H2 and 40mU of a107V SEL29 rnase H2), and from 0.0 cycles with WT p.a. rnase H2 to 11.5 cycles with Q48R SEL29 rnase H2 (for 5mU of WT rnase H2 and 100mU of Q48R SEL29 rnase H2). Mismatch discrimination ΔC of rs3117947SNP _q Quantification increased from 10.7 cycles with WT P.a rnase H2 to 22.3 cycles with a107V SEL29 rnase H2 (for 5mU of WT rnase H2 and 40mU of a107V SEL29 rnase H2), and from 10.7 cycles with wtp.a. rnase H2 to 20.4 cycles with Q48R SEL29 rnase H2 (for 5mU of WT rnase H2 and 100mU of Q48R SEL29 rnase H2). These mismatch discrimination increases for Q48R SEL29 and a107V SEL29 rnase H2 are significantly greater than for background SEL29 rnase H2. Thus, Q48R SEL29 and a107V SEL29 rnase H2 can enhance mismatch discrimination when the mismatch is located 3' of the RNA base.

Example 6. Use of Q48R SEL29 and A107V SEL29 hybrid RNase H2 proteins in the LAMP reaction.

This example outlines a method for demonstrating the use of hybrid rnase H2 protein in LAMP reactions to reduce primer dimer formation.

To evaluate the function of rhPrimers on LAMP protocols comprising different rnase H2 proteins (WT or hybrid rnase H2 proteins), three assays can be designed using: (1) an unmodified control primer; "Gen1" rDDDDMx primer, wherein "r" is an RNA base, "D" is a DNA base, "m" is a mismatch, and "x" is a C3 spacer; and "Gen2" rDxxDM primers. Details of the assays used for the evaluation are detailed in tables 14, 15 and 16. The LAMP reaction using each type of primer will be maintained at 25℃for 0 or 2 hours prior to testing. This will allow the formation of primer dimer products. After maintaining room temperature, all reactions were run in BioRad CFX384 or Roche LightCycler480 at 65 ℃ for 2 hours. Signal generation in all of these reactions will be performed using 1 XEvaGreen added to the reaction (see: biotechnology Letters, 12 months 2007, 29, 12 th edition, pages 1939-1946).

Table 14: controlled unmodified primer design.

DNA bases are capitalized; RNA bases are lowercase. F=6-carboxyfluorescein; q=iowa Black ^TM FQ fluorescence quenchers.

Table 15: gen1 primer design.

DNA bases are capitalized; RNA bases are lowercase. F=6-carboxyfluorescein; q=iowa Black ^TM FQ fluorescence quenchers. X=c3 spacers.

Table 16: and (5) designing a GEN2 primer.

DNA bases are capitalized; RNA bases are lowercase. F=6-carboxyfluorescein; q=iowa Black ^TM FQ fluorescence quenchers. X=c3 spacer

Each assay can be performed as follows: the sample will be Coriell gDNA or lambda phage genomic DNA. Each assay condition can be run in triplicate, with samples input either 5ng lambda phage genomic DNA or 20ng human genomic DNA. For each assay, the reaction can be run using unmodified primers with intercalating dye (e.g., evaGreen) and cleavable, blocking primers with intercalating dye. For each assay, the reaction will be run using zero or titrated levels of rnase H2 (WT or mixed rnase H2 protein).

Each assay will be run in triplicate. By comparing the length of time required for signal formation products to form, the unmodified LAMP assay and the modified LAMP assay will be compared. It is expected that the time for these products to be produced by the hybrid RNase H2 protein in the LAMP reaction will be significantly later than the reaction containing the conventional wild-type RNase H2 protein.

25. Mu.L EvaGreen reaction mixture will include:

12.5. Mu.L of 2 Xmaster mix (1X 20mM Tris pH 8.8@25 ℃,10mM KCl,10mM (NH) ₄ ) ₂ SO ₄ ，8mM MgSO ₄ ，0.01％ Tween-20，1.4mM dNTP)

1.6. Mu.M FIP primer

1.6 mu.M BIP primer

1x EvaGreen dye

0.2 mu M F3 primer

0.2 mu M B3 primer

8U BST DNA polymerase (New England Biolabs: https:// www.neb.com/products/m 0275-BST-DNA-polymerase-large-fragment)

1. Mu.L of hybrid RNase H2 protein (buffer D will be used for RNase-free H2 control)

Nuclease-free water to 25uL

mu.L of sample (10 ng/. Mu.L of human gDNA or 2.5 ng/. Mu.L of lambda.genomic DNA)

Example 7. Q48R SEL29 RNase H2 improved the quality of the NGS library compared to wild-type P.a.RNase H2 in a multiplex rhPCR amplicon sequencing workflow using the 177-plex detection group.

The modified rhAmpSeq scheme is suitable for low-frequency variation detection. This protocol uses both high fidelity DNA polymerase to reduce amplification errors and Unique Molecular Identifiers (UMIs) for error correction. Q48R SEL29 rnase H2 was compared to wild-type P.a rnase H2 in the system to determine if dimer formation was reduced.

There were two PCR cycling steps, each followed by SPRI (solid phase reversible immobilization (Solid Phase Reversible Immobilization), beckman Coulter Life sciences, indianapolis, ind.) clean-up. The purpose of the first PCR step (PCR 1) was to incorporate 6 nucleotide degenerate UMI on each side of the target amplicon. This step also includes the use of 3' -blocked primers and requires rnase H2 to cleave the blocker and allow high fidelity DNA polymerase to extend and amplify each target. The target-specific assay set contained 177 proprietary primer pairs, with approximately 20% of the total reads being primer dimers when wild-type p.aarnase H2 was present in PCR 1. This group generated a large number of primer dimers, making it the best choice for testing Q48R SEL29 rnase H2.

Various 10X titrations of Q48R SEL29 RNase H2 and wild type P.a. RNase H2 (87.5 mU/uL, 175mU/uL and 350 mU/uL) were prepared in RNase H2 storage buffer (Integrated DNAtechnologies, coralville, IA). The final volume of each PCR1 reaction was 20. Mu.L, containing 10nM (200 fmol) of each forward and reverse primer, 0.03U/. Mu. L Phusion Hot Start IIDNA polymerase (Thermo Fisher Scientific, waltham, mass.), 20ng genomic cell line DNA (cell lines NA24385, coriell Institute for Medical Research, camden, NJ) and 1 Xfinal concentration of RNase H2 in proprietary high-fidelity polymerase buffer. Thermal cycling T100 thermal cycler (Bio-Hercules, CA), the cycle conditions are listed in table 17. SPRI clean-up was performed immediately after PCR1 thermal cycling. 1.25X (25. Mu.L) AMPure magnetic beads (Beckman Coulter Life Sciences, indianapolis, ind.) were added to each well and mixed well. Plates were incubated for 5 minutes at room temperature on a bench and then 5 minutes on a magnet. The supernatant was discarded and the library was washed twice with 80% ethanol. The sample was eluted in 22. Mu.L of IDTE pH 7.5 (IDT, coralville, IA). 20. Mu.L of the eluted product was used as input for PCR 2.

TABLE 17 PCR1 cycle conditions.

The purpose of the second PCR step (PCR 2) is to amplify the PCR 1 product and add a unique sample strandThe primer sequences were used for pooling and sequencing purposes. PCR 2 does not require the use of RNase H2. The PCR 2 reaction was performed in a final volume of 50. Mu.L. The 2X high fidelity polymerase buffer used in PCR 1 was added to each eluted sample of PCR 1 at a final concentration of 1X. Each reaction also had a unique combination of i5 and i7 rhampheq index primers (IDTs), each 500nM. Thermal cycling T100 thermal cycler (Bio-Hercules, CA), the cycle conditions are listed in table 18. SPRI clean-up was performed immediately after PCR 2, as in the procedure outlined above, except that the AMPure microbead concentration was varied and it was used at 0.9X after PCR 2. The sample was eluted in 22. Mu.L of IDTE pH 7.5 (IDT, coralville, IA). 20. Mu.L of the eluted product was taken and stored at-20℃until sequencing.

TABLE 18 PCR 2 cycle conditions.

The resulting libraries were pooled in equal volumes for sequencing (5 uL). The pool was quantified using the Qubit dsDNAHS assay kit (Thermo Fisher) and diluted to a final concentration of 4 nM. Equal amounts of library pools were mixed with 0.2N NaOH to denature the library. The reaction was then diluted to a final concentration of 8pM, which contained 2.5% PhiX (Illumina, san Diego, calif.) incorporation. The reaction was loaded onto a MiSeq 300 cycling kit (Illumina, san Diego, calif.) and run (cluster density: 1002.+ -. 34; Q30:93.13%). The results were run through an internal proprietary IDT rhampeq VII bioinformatics analysis tube.

For purposes of this disclosure, calculations and terms related to the following results are listed herein. The amount of primer dimer generated during amplification of the sequencing library is defined by the dimer rate. The rhampheq VII tube calculates dimer rate by dividing the total count of identified dimers by the number of reads that passed QC (total number of reads that passed the purity filter). The mapping rate is calculated by: the total number of reads mapped is divided by the number of reads passed by QC. The mid-target rate was calculated by: the total number of reads for the mid target is divided by the number of reads passed by QC. Total amplicon uniformity or amplicon uniformity ∈ 0.2X is calculated as the percentage of normal amplicons with greater than or equal to 0.2 times the average coverage of the amplicons. Loss rate or uniformity was calculated to be less than 0.05X as the percentage of normal amplicon that is less than 0.05 times the average coverage of amplicon. Amplicon uniformity distribution the coverage of each amplicon was compared to the average amplicon coverage for a given sample and plotted over the following range: 0.1-0.2X, 0.2-0.5X, 0.5-1.5X, 1.5-2.5X and 2.5-5X.

The rhampheq VII channel also identified primers that contributed to the formation of each primer dimer. Normalized dimer counts reflect the percentage of each primer dimer to total dimer rate and are calculated using the following formula:

For all concentrations tested, Q48R SEL29 rnase H2 reduced the dimer rate compared to wild-type P.a rnase H2, while also improving the mapping and targeting rates. The library generated at the lowest concentration (8.75 mU/. Mu.L) of Q48R SEL29 RNase H2 had 4% dimer compared to the control wild-type P.a RNase H2 that produced 11% dimer (FIG. 1, panel A).

The mapping and targeting rates of the library generated using the lowest concentration of Q48R SEL29 rnase H2 (8.75 mU/μl) increased to 96% compared to 88% for wild-type P.a rnase H2 (fig. 1, panels B and C).

The dimer rates may be broken down into individual primer dimers produced and then normalized to determine the contribution of each primer dimer to the total dimer rate, as described above (normalized dimer counts). Q48RSEL29 rnase H2 reduced the number of most primer dimers by half. For example, in the library generated using the lowest concentration of Q48R Mut 29 RNase H2 (8.75 mU/. Mu.L), the front dimer (NOTCH1_19.GAP95.9623.relay.t0_FOR: NOTCH1_9.GA104.1246.relay.t0_REV) identified in all sample libraries was only 1.23% of the total dimer rate, compared to 2.83% in the case of wild-type P.a RNase H2. Some dimers were reduced by more than half in the following cases: it was not determined that the TERT 15.GAP27.511.relax.t0_REV Q48R SEL29RNA enzyme H2 library produced a 75% reduction in dimer compared to wild-type P.a rnase H2 of 0.11% where the dimer accounted for 0.47% of the total dimer rate (table 19 and fig. 2).

Table 19 normalized dimer counts identified/each primer pair described in example 1.

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The ability of Q48R SEL29 rnase H2 to reduce dimer rates does not affect library yield, overall uniformity and loss rate or uniform distribution index. Q48R SEL29 rnase H2 produced similar library yields at all titration concentrations tested as demonstrated by amplicon average coverage compared to wild-type P.a rnase H2 enzyme (fig. 3, panel a). Furthermore, overall amplicon uniformity was ≡0.2X and amplicon loss rate (amplicon uniformity +.0.05X) were comparable between Q48R SEL29 rnase H2 and wild-type p.a. enzyme at all titration concentrations tested (fig. 3, panels B and C). Furthermore, the uniformity profile between Q48RSEL29 and wild-type P.a rnase H2 appears similar (fig. 4). During library generation of the rhampeq workflow, Q48R SEL29 rnase H2 reduced primer dimer formation compared to standard wild-type p.a. rnase H2 without altering other important sequencing metrics.

Taken together, these data demonstrate that Q48R SEL29 rnase H2 use of high fidelity DNA polymerase in high fidelity buffers improved generation of multiple next generation sequencing libraries.

Example 8 exemplary amino acid and nucleic acid sequences encoding an RNase H2 protein.

Exemplary amino acid and nucleic acid sequences encoding the rnase H2 protein are shown below.

Table 20. Nucleic acid sequences encoding RNase H2 proteins previously described.

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¹ The location of the mutation is shown in bold and underlined.Plasmid extension (His) ₆ The tag is shown in italics, including the stop codon.

TABLE 21 amino acid sequence of RNase H2 protein lacking the C-terminal extension sequence.

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¹ The location of the mutation is shown in bold and underlined.

Table 22. Nucleic acid sequences encoding RNase H2 proteins lacking additional C-terminal extension sequences.

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¹ The location of the mutation is shown in bold and underlined. The stop codon is shown in italics.

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。

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually indicated to be incorporated by reference and were set forth in its entirety herein. Each reference is individually and specifically indicated to be incorporated by reference and set forth in its entirety herein.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A hybrid rnase H2 protein comprising amino acid sequence fragments from a thermococcus deep sea (p.a.), a rhodococcus costa (t.kod), and a pyrococcus intensiviss organism.

2. The hybrid rnase H2 protein of claim 1, wherein the hybrid rnase H2 protein comprises amino acid residues 26-40 and residues 100-120 of t.kod rnase H2.

3. The hybrid rnase H2 protein of claim 1, wherein the hybrid rnase H2 protein is selected from the group consisting of SEQ ID NOs 2 and 3.

4. The hybrid rnase H2 protein of claim 1, wherein the hybrid rnase H2 protein is selected from SEQ ID NOs 14-20.

5. A recombinant nucleic acid encoding any of the hybrid rnase H2 proteins of claims 1-4.

6. A method of performing primer extension, the method comprising: contacting the hybrid rnase H2 protein of any one of claims 1-4 with a primer, a polynucleotide template, a nucleoside triphosphate, and a DNA polymerase under conditions suitable for a primer extension method, thereby producing an extended primer.

7. The method of claim 6, wherein the DNA polymerase comprises a high fidelity archaea DNA polymerase.

8. The method of claim 6, wherein the primer comprises a blocked cleavable primer.

9. The method of claim 8, wherein the primer extension method comprises a method of performing a Polymerase Chain Reaction (PCR).

10. The method of claim 9, wherein the method of performing PCR improves mismatch discrimination in a primer:polynucleotide hybrid formed between the primer and the polynucleotide template.

11. The method of claim 10, wherein the improvement in mismatch discrimination comprises an improvement in 3' -mismatch discrimination.

12. A reaction mixture comprising the hybrid rnase H2 protein of claims 1-4, at least one primer, a polynucleotide template, a nucleoside triphosphate, and a DNA polymerase.

13. The reaction mixture of claim 12, wherein the DNA polymerase comprises a high fidelity archaea DNA polymerase.

14. The reaction mixture of claim 12 or 13, wherein the at least one primer comprises a blocked cleavable primer.

15. A method of performing rhPCR, the method comprising primer extension with the hybrid rnase H2 of any one of claims 1-4 and a primer.

16. The method of performing rhPCR of claim 15, comprising primer extension using a high-fidelity archaea DNA polymerase.

17. The method of claim 15 or 16, wherein the hybrid rnase H2 enzyme is reversibly inactivated by chemical modification, aptamer, or blocking antibody.

18. The method of claim 17, wherein a blocking group is attached to the 3' terminal nucleotide of the primer.

19. The method of claim 18, wherein the blocking group is attached 5 'to the 3' terminal residue and inhibits the primer from acting as a template for DNA synthesis.

20. The method of claim 19, wherein the blocking group comprises one or more abasic residues.

21. The method of claim 24, wherein the one or more abasic residues is a C3 spacer.

22. The method of any one of claims 18-21, wherein the blocking group comprises a member selected from the group consisting of: RDDDDx, RDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD, wherein R is an RNA residue, D is a DNA residue, M is a mismatch residue, and x is a C3 spacer or other means of preventing extension of the DNA polymerase.

23. The method of any one of claims 18-22, wherein the blocking group comprises a label that allows detection of an extension amplification reaction.

24. The method of claim 23, wherein a label allowing detection of the amplification reaction is attached to the oligonucleotide primer 3' to the cleavage site.

25. The method of claim 24, wherein the label is a fluorophore.

26. The method of claim 23, wherein the label is a mass tag for detecting an amplification reaction by mass spectrometry.

27. The method of any one of claims 14-26, wherein the cleavage domain of the blocked cleavable primer comprises one or more of the following moieties: DNA residues, abasic residues, modified nucleosides or modified internucleotide linkages.

28. The method of any one of claims 14-26, wherein the cleavage domain comprises a single RNA residue.

29. The method of any one of claims 14-26, wherein the cleavage domain comprises two adjacent RNA residues.

30. The method of any one of claims 14-26, wherein the cleavage domain comprises a contiguous sequence of three or more RNA residues.

31. The method of any one of claims 14-26, wherein the cleavage domain lacks RNA residues.

32. The method of any one of claims 14-26, wherein the cleavage domain comprises one or more 2' -modified nucleosides.

33. The method of claim 32, 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' -thio nucleosides.

34. The method of claim 32, wherein the one or more 2 '-modified nucleosides is a 2' -O-methyl RNA nucleoside.

35. The method of claim 32, wherein the one or more 2 '-modified nucleosides is a 2' -fluoronucleoside.

36. A method of amplifying a target DNA sequence, the method comprising:

(a) Providing a reaction mixture comprising:

(i) An oligonucleotide primer having a cleavage domain cleavable by an RNAse H2 enzyme, located 5 'of a blocking group attached at or near the 3' end of the oligonucleotide primer, wherein the blocking group prevents primer extension and/or inhibits the oligonucleotide primer from acting as a template for DNA synthesis,

(ii) Sample nucleic acid which may or may not be a target sequence,

(iii) DNA polymerase, and

(iv) The hybrid rnase H2 protein of claims 1-4;

(b) Hybridizing the oligonucleotide primer to the target DNA sequence to form a double stranded substrate;

(c) Cleaving 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.

37. The method of claim 36, wherein the DNA polymerase is a hi-fi archaea DNA polymerase.

38. The method of any one of claims 36 or 37, wherein the rnase H2 protein is reversibly inactivated by chemical modification, aptamer, or blocking antibody.

39. The method of any one of claims 36-38, wherein the blocking group is attached to the 3' terminal nucleotide of the oligonucleotide primer.

40. The method of any one of claims 36-38, wherein the blocking group is attached 5 'to the 3' terminal residue and inhibits the oligonucleotide primer from acting as a template for DNA synthesis.

41. The method of any one of claims 36-40, wherein the blocking group comprises one or more abasic residues.

42. The method of claim 41, wherein the one or more abasic residues are C3 spacers.

43. The method of any one of claims 40 or 41, wherein the blocking group comprises a member selected from the group consisting of: RDDDDx, RDDDDMx, RDxxD, RDxxDM, RDDDDxxD, RDDDDxxDM and DxxD, wherein R is an RNA residue, D is a DNA residue, M is a mismatch residue, and x is a C3 spacer or other means of blocking extension of the DNA polymerase.

44. The method of any one of claims 36-43, wherein the blocking group comprises a label that allows detection of an extension amplification reaction.

45. The method of any one of claims 36-44, further comprising a label that allows detection of an amplification reaction, wherein the label is attached to an oligonucleotide primer 3' to the cleavage site.

46. The method of claim 45, wherein the label is a fluorophore.

47. The method of claim 45, wherein the label is a mass tag for detecting an amplification reaction by mass spectrometry.

48. The method of any one of claims 36-47, wherein the cleavage domain comprises one or more of the following moieties: DNA residues, abasic residues, modified nucleosides or modified internucleotide linkages.

49. The method of any one of claims 36-48, wherein the cleavage domain comprises a single RNA residue.

50. The method of any one of claims 36-48, wherein the cleavage domain comprises two adjacent RNA residues.

51. The method of any one of claims 36-48, wherein the cleavage domain comprises a contiguous sequence of three or more RNA residues.

52. The method of any one of claims 36-48, wherein the cleavage domain comprises one or more 2' -modified nucleosides.

53. The method of claim 52, 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' -thio nucleosides.

54. The method of claim 52, wherein the one or more 2 '-modified nucleosides is a 2' -O-methyl RNA nucleoside.

55. The method of claim 52, wherein the one or more 2 '-modified nucleosides is a 2' -fluoronucleoside.

56. The method of any one of claims 36-55, wherein the sequence flanking the cleavage site comprises one or more nuclease-resistant internucleotide linkages.

57. The method of claim 56, wherein said nuclease-resistant linkage is phosphorothioate.

58. A kit for producing an extended primer, the kit comprising at least one container providing the hybrid rnase H2 protein of claims 1-4.

59. The kit of claim 58, further comprising one or more additional containers selected from the group consisting of: (a) Providing a container of primers which hybridize to a predetermined polynucleotide template under primer extension conditions; (b) providing a container of nucleoside triphosphates; (c) Providing a container of buffer suitable for primer extension and (d) a DNA polymerase.

60. The kit of claim 59, wherein the DNA polymerase comprises a Hi-Fi archaea DNA polymerase.

61. The kit of any one of claims 58-60, further comprising one or more additional containers selected from the group consisting of: a container comprising blocked cleavable primers.

62. A kit for performing amplification of a target DNA sequence, the kit comprising a reaction buffer comprising the rnase H2 of claims 1-4 and a hi-fi archaea DNA polymerase.

63. The kit of claim 62, further comprising one or more oligonucleotide primers, wherein at least one oligonucleotide primer has a cleavage domain cleavable by an rnase H2 enzyme, located 5 'of a blocking group attached at or near the 3' end of the oligonucleotide primer, wherein the blocking group prevents primer extension and/or inhibits the oligonucleotide primer from acting as a template for DNA synthesis.

64. The kit of claim 63, wherein 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 moiety that blocks extension of the DNA polymerase.

65. A method of preparing an amplicon library of a template nucleic acid, the method comprising:

forming a mixture comprising:

a population of nucleic acids;

at least a blocked cleavable primer;

hybrid rnase H2 protein;

dNTP；

a DNA polymerase; and

the buffer solution is used for the preparation of the liquid,

wherein hybridization duplex is formed between the at least blocked cleavable primer and the population of nucleic acids in the mixture;

cleaving the at least one blocked cleavable primer with a hybrid mutant rnase H2 protein to produce at least one active primer capable of primer extension by the DNA polymerase; and

extending the at least one active primer in the buffer with the DNA polymerase under conditions that allow for amplification of one or more template nucleic acids from the population of nucleic acids, thereby generating amplicons of the template nucleic acids.

66. The method of claim 65, wherein the hybrid RNase H2 protein is selected from Q48RSEL29 (SEQ ID NO: 18) or others.

67. The method of claim 65, wherein the DNA polymerase is KOD DNA polymerase, or other high fidelity archaea DNA polymerase.

68. The method of claim 65, wherein the buffer is a KOD DNA polymerase buffer, or another high fidelity archaea DNA polymerase buffer.

69. A method of performing massively parallel sequencing, the method comprising:

preparing a population of template nucleic acid libraries comprising:

PCR was performed with a mixture comprising:

nucleic acid population:

hybrid rnase H2 muteins:

at least one blocked cleavable primer:

DNA polymerase:

dNTP; and

a buffer; and

sequencing a plurality of desired template nucleic acids from the template nucleic acid library population.

70. The method of claim 69, wherein the hybrid RNase H2 protein is selected from Q48RSEL29 (SEQ ID NO: 18) or others.

71. A method of detecting a SNP-containing nucleic acid template from a nucleic acid template amplicon library, the method comprising:

forming a mixture comprising:

a library of nucleic acid template amplicons;

at least a blocked cleavable primer;

hybrid rnase H2 protein;

dNTP；

a DNA polymerase; and

the buffer solution is used for the preparation of the liquid,

wherein hybridization duplex is formed between the at least blocked cleavable primer and the SNP containing nucleic acid template in the library of nucleic acid template amplicons in the mixture;

cleaving at least one blocked cleavable primer of said hybrid duplex with said hybrid rnase H2 protein to generate at least one active primer capable of primer extension of said hybrid duplex by said DNA polymerase; and

Extending the at least one active primer in the hybridization duplex with the DNA polymerase in the buffer under conditions that allow amplification of one or more template nucleic acids from the nucleic acid template amplicon library, thereby detecting the SNP containing nucleic acid template.

72. The method of claim 71, wherein the hybrid RNase H2 protein is selected from Q48RSEL29 (SEQ ID NO: 18) or others.

73. A method of performing a loop-mediated amplification reaction, the method comprising:

forming a mixture comprising:

a nucleic acid template;

four blocked cleavable primers, wherein the blocked cleavable primers form a duplex with a nucleic acid template that is a substrate for an rnase H2 protein;

an rnase H2 protein, wherein the rnase H2 protein is selected from Q48R SEL29 (SEQ ID No.: 18) or others;

a DNA polymerase protein;

dNTP; and

a buffer; and

isothermal amplification cycles were performed with the mixture.

74. A product mixture produced using the method of any one of claims 6, 15, 36, 69, 71 and 73 with Q48R SEL29 rnase H2 (SEQ ID NO: 18), wherein the product mixture produced therefrom comprises a reduced population of primer dimer species relative to a product mixture produced with wild-type p.a.rnase H2 (SEQ ID NO: 1).

75. A method of rhPCR assay with reduced primer dimer formation, the method comprising primer extension with Q48R SEL29 rnase H2 (SEQ ID NO: 18), wherein reduced primer dimer formation corresponds to the amount of primer dimer reduced during the rhPCR assay with Q48R SEL29 rnase H2 (SEQ ID NO: 18) when compared to the rhPCR assay with wild-type p.a. rnase H2 (SEQ ID NO: 1).

76. A method of rhPCR assay with improved mapping and mid-target rate for a desired product, the method comprising primer extension with Q48R SEL29 rnase H2 (SEQ ID NO: 18), wherein the improved mapping and mid-target rate corresponds to increased mapping and mid-target amplification of the desired product formed during the rhPCR assay with Q48R SEL29 rnase H2 (SEQ ID NO: 18) when compared to the rhPCR assay with wild-type p.a. rnase H2 (SEQ ID NO: 1).