Nucleic acid amplification methods and compositions

Abstract

Methods are provided for nucleic acid amplification using primers that contain DNA oligonucleotide segments and at least one ribonucleotide. In PCR reactions, this increases the amount of amplified product with each amplification cycle, resulting in greater sensitivity and speed than conventional PCR. In isothermal reactions, this reduces the time required to detect the target. Also provided are methods for monitoring amplification reactions using probes that contain ribonucleotides, without relying on the 5'-nuclease activity of DNA polymerase.

Description

[Technical field]

[0001] The methods and compositions described herein relate generally to the field of nucleic acid amplification and detection. In particular, methods and compositions for increasing amplification efficiency are described herein. [Background technology]

[0002] A wide variety of nucleic acid amplification techniques are available, many of which are used to perform sensitive diagnostic assays based on nucleic acid detection.

[0003] Polymerase chain reaction (PCR) remains the most widely used DNA amplification and quantification method. However, PCR generally has some limitations. PCR amplification can increase the amount of target sequence less than two-fold with each cycle. PCR is still relatively slow, and PCR sensitivity is limited. Similarly, isothermal amplification methods rely on the extension of DNA primers. Once extended, the primers are not further used to initiate the next round of extension.

[0004] Primers are widely used in various methods of nucleic acid amplification. They are generally DNA oligonucleotides designed to hybridize to a target nucleic acid and are extended by a polymerase. Once the primer is extended, it is incorporated into the extended amplicon and is no longer used for amplification. The use of standard primers alone places an upper limit on the efficiency of PCR, limiting the rate of amplification and detection of nucleic acids.

[0005] The PCR primers used in PCR amplification are usually all deoxynucleotides, because DNA polymerases used in PCR do not normally use ribonucleotides as templates. Even some polymerases may be able to copy ribonucleotides (e.g., reverse transcriptase activity), but the general belief is that ribonucleotides in primers are avoided because substituting ribonucleotides for deoxynucleotides usually increases primer cost, decreases primer stability, and decreases PCR efficiency.

[0006] Efforts to improve nucleic acid amplification efficiency have focused on improving primer design, polymerase function, and reaction conditions. For example, U.S. Patent No. 8,252,558 teaches a form of nested PCR. U.S. Patent Publication No. 2019 / 0203266A1 discloses a method for improving PCR efficiency through primer design and the use of strand-displacing DNA polymerases. Summary of the Invention

[0007] Described herein are methods, compositions and systems based on the use of primers that contain at least one ribonucleotide, which are designed to initiate multiple rounds of template-based chain extension in the presence of enzymes such as RNase H2 and polymerase.

[0008] In one embodiment of the present disclosure, a nucleic acid primer set is provided. The primer set includes a first primer having a 3' end and a 5' end for amplifying a target nucleic acid in a sample, the target nucleic acid including a first template strand. The first primer includes a first segment of a DNA oligonucleotide and a second segment of a DNA oligonucleotide, the first segment being located on the 5' side of the second segment, and the first segment and the second segment being linked by at least one first ribonucleotide. At least a portion of the second segment can specifically hybridize to the first template strand to initiate nucleotide polymerization and generate an extension product complementary to the first template strand.

[0009] In some embodiments, at least one first ribonucleotide of the first primer comprises two or more ribonucleotides connected contiguously, In some embodiments, at least one first ribonucleotide of the first primer comprises two or more ribonucleotides spaced apart, In some embodiments, at least one first ribonucleotide of the first primer consists of a single ribonucleotide.

[0010] In some embodiments, both the first and second segments of the first primer specifically hybridize to the first template strand under a given condition. In some embodiments, the first segment does not specifically hybridize to the first template strand, but the second segment specifically hybridizes to the first template strand. In some embodiments, only a portion of the second segment (not the entire second segment) specifically hybridizes to the first template strand.

[0011] In some embodiments, the target nucleic acid further comprises a second template strand complementary to the first template strand, and the primer set further comprises a second primer capable of specifically hybridizing to the second template strand. In certain embodiments, the second primer has a 3' end and a 5' end, and comprises a third segment of a DNA oligonucleotide and a fourth segment of a DNA oligonucleotide, the third segment being located at the 5' side of the fourth segment, and the third segment and the fourth segment being linked. At least one second ribonucleotide allows at least the fourth segment to specifically hybridize to the second template strand.

[0012] In another aspect of the disclosure, a method for amplifying a target nucleic acid in a sample is provided, the target nucleic acid comprising a first template strand, the method comprising: (a) contacting the sample with a first primer having a 3' end and a 5' end, the first primer comprising a first oligonucleotide segment and a second oligonucleotide segment, under conditions in which at least a portion of the second oligonucleotide segment anneals to the first template strand, the first oligonucleotide segment being 5' to the second oligonucleotide segment and linked to the second oligonucleotide segment by at least one first ribonucleotide, (b) extending the first primer using the first template strand as a template to generate a first amplicon complementary to the first template strand, and (c) cleaving the first primer at a position 5' immediately preceding the at least one first ribonucleotide, the cleavage can be accomplished using RNase H2.

[0013] In some embodiments, the first segment anneals to the first template strand under the conditions described above, and the method further comprises extending the first segment of the first primer using the first template strand as a template while displacing the first amplicon from the first template strand to generate a second amplicon that is complementary to the first template strand.

[0014] In some embodiments, if the first segment of the first primer does not anneal to the first template strand under the above conditions, the method further comprises the step of: The method includes the steps of separating the first amplicon from the first template strand prior to cleaving in (c), generating a nucleic acid strand complementary to the first amplicon using a second primer, the new nucleic acid strand comprising a segment complementary to the first segment of the first primer, and extending the first segment of the first primer using the new nucleic acid strand as a template after cleaving in (c) to generate a third amplicon complementary to the new nucleic acid strand while displacing the first amplicon from the new nucleic acid strand, wherein in some of these embodiments, at least a portion 3' of the at least one first ribonucleotide and distal to the 3' end of the first primer does not specifically hybridize to the first template strand.

[0015] In some embodiments, the target nucleic acid further comprises a second template strand complementary to the first template strand, and the method further comprises (d) contacting the sample with a second primer comprising a third segment of a DNA oligonucleotide and a fourth segment of a DNA oligonucleotide, the third segment being 5' to the fourth segment and linked to the fourth segment by at least one second ribonucleotide, at least a portion of the fourth segment anneals to the second template strand under conditions, (e) extending the second primer using the second template strand as a template to generate a second amplicon complementary to the second template strand, and (f) cleaving the second primer at a position 5' immediately preceding the at least one second ribonucleotide.

[0016] In some embodiments, the first segment and the second segment both anneal to the first template strand under conditions, and the target nucleic acid further comprises a second template strand complementary to the first template strand, and the method further comprises (d) contacting the sample with a second primer comprising a third segment that is a DNA oligonucleotide and a fourth segment that is a DNA oligonucleotide, wherein the third segment is 5' to the fourth segment and is linked to the fourth segment by at least one second ribonucleotide, and wherein at least a portion of the fourth segment anneals to the second template strand under the conditions. (e) extending the second primer using the second template strand as a template to generate a second amplicon complementary to the second template strand; (f) cleaving the second primer at a position 5' immediately preceding the at least one second ribonucleotide; (g) extending the first segment of the first primer using the first template strand as a template to generate a third amplicon complementary to the first template strand while displacing the first amplicon from the first template strand to generate a fourth amplicon complementary to the first template strand; and (h) extending the second primer using the second template strand as a template while displacing the third amplicon from the second template strand to form a second template strand. In some of these embodiments, the method may further include (h) using another second primer to generate a fifth amplicon complementary to the first amplicon. The method may include using another first primer to generate a sixth amplicon complementary to the second amplicon. Thermal cycling may be performed to further amplify the amplification products obtained in the process. For example, the temperature is increased to separate the generated double stranded amplicons, and the temperature is decreased to allow the first and second primers to further anneal to the separated amplicons.

[0017] In some embodiments of the methods described herein, the first primer is immobilized on a solid surface or in a matrix.

[0018] In some embodiments of the methods described herein, the first template strand is RNA.

[0019] In some embodiments, the method further comprises using the first segment cleaved from the first primer as a primer to initiate nucleotide polymerization using a second target nucleic acid.

[0020] In a further embodiment, a method for detecting a nucleotide variation of a target nucleic acid in a sample is provided. The sample comprises a first double-stranded DNA and a second double-stranded DNA, and the second double-stranded DNA is different from the first double-stranded DNA at at least one difference position. The method includes:

[0021] (a) contacting the sample with a first primer having a 3' end and a 5' end, the first primer comprising a first oligonucleotide segment and a second oligonucleotide segment, wherein the first oligonucleotide segment is 5' to the second oligonucleotide segment and is bound to the second oligonucleotide segment by at least one first ribonucleotide under conditions in which the first primer anneals to both the first template strand of the first double-stranded DNA and the first template strand of the second double-stranded DNA, but the second segment of the first primer does not anneal by itself to either the first template strand of the first double-stranded DNA or the first template strand of the second double-stranded DNA, and the ribonucleotide of the first primer is at or near the mutation position, and the first primer is positioned such that it perfectly matches the first template strand of the first double-stranded DNA but not perfectly matches the first template strand of the second double-stranded DNA.

[0022] (b) using the first template strand of the first double-stranded DNA as a template to generate a first amplicon by extending a first primer hybridized on the first template strand of the first double-stranded DNA, and using the first template strand of the second double-stranded DNA as a template to generate a second amplicon by extending the first primer hybridized on the first template strand of the second double-stranded DNA.

[0023] (c) cleaving the first primer hybridized to the first template strand of the first double-stranded DNA with an RNase at a position 5' immediately preceding the first ribonucleotide of at least one of the first double-stranded DNAs, thereby generating a 5' segment cleaved from the first primer hybridized to the first template strand of the first double-stranded DNA, while not cleaving the first primer hybridized to the first template strand of the second double-stranded DNA.

[0024] and (d) performing PCR to further amplify the first and second double-stranded DNAs in the presence of RNase, and distinguishing the first and second double-stranded DNAs by the amount of each amplicon product.

[0025] In a further aspect, a method for amplifying a target nucleic acid in a sample is provided, the target nucleic acid comprising a first template strand. The method includes:

[0026] The method includes the steps of: (a) contacting the sample with a first primer having a 3' end and a 5' end, the first primer comprising a first oligonucleotide segment and a second oligonucleotide segment, the first oligonucleotide segment being 5' to the second oligonucleotide segment and linked to the second oligonucleotide segment by at least one first ribonucleotide such that at least a portion of the second segment is capable of annealing to a first template strand.

[0027] (b) extending a first primer using the first template strand as a template to generate a first extension product that is complementary to the first template strand; (c) performing PCR, each amplicon comprising a first primer, to generate multiple copies of said first extension product; (d) cleaving the first primer in the amplicon of the first extension product at a position 5' immediately preceding the at least one first ribonucleotide to generate a cleaved portion of the replicated copy; (e) annealing or ligating the cut portion of the replicated copy to another nucleic acid strand.

[0028] In a further aspect, a molecular probe for detecting amplification of a nucleic acid is provided. The probe comprises a quencher portion, a fluorophore portion, and a linker portion connecting the quencher portion and the fluorophore portion, the linker portion comprising a plurality of deoxyribonucleotides and ribonucleotides, and when the linker is intact, no fluorescence is emitted by the fluorophore. Thus, a method for detecting an amplicon product in an amplification system is provided. The method comprises the steps of specifically hybridizing the molecular probe to an amplicon product in an amplification reaction system, cleaving ribonucleotides in the molecular probe, and detecting the fluorescence emitted by the fluorophore portion of the molecular probe.

[0029] Various embodiments contemplated herein may include, but are not necessarily limited to, one or more of the following. [Brief description of the drawings]

[0030] FIG. 1 is a schematic diagram showing ribonucleotide-containing primer amplification of a template followed by cleavage by RNase H2, ready for a second round of extension, according to an embodiment of the invention.

[0031] FIG. 2 is a schematic diagram showing the extension and displacement of an amplicon using a primer containing two spaced apart ribonucleotides in the presence of a strand-displacing DNA polymerase and RNase H2, according to an embodiment of the invention.

[0032] FIG. 3 is a schematic diagram showing the extension and displacement of an amplicon using a primer containing multiple consecutive ribonucleotides in the presence of a strand-displacing DNA polymerase and RNase, according to an embodiment of the invention.

[0033] FIG. 4 is a schematic diagram illustrating the generation of a DNA template by reverse transcription of an RNA template with a ribonucleotide-containing primer in the presence of a reverse transcriptase with RNase H activity, or a reverse transcriptase and RNase H, according to an embodiment of the invention.

[0034] 5 is a schematic diagram showing the amplification of a whole genome, whole transcriptome, or multiple target DNA by using multiple primers, each containing a ribonucleotide, that anneal to a template, are extended by a polymerase, and are then cleaved by RNase H2, and further extended by the polymerase, according to an embodiment of the invention.

[0035] 6 is a schematic diagram showing the amplification of whole genomes, whole transcriptomes, or multiple target DNAs using multiple primers in the presence of NTPs, where ribonucleotides are incorporated by a polymerase and then cleaved and further extended by RNase H2. According to an embodiment of the invention, the primers optionally comprise at least ribonucleotides. FIG. 7 is a schematic diagram showing the generation of two amplicons from one ribonucleotide containing primer that includes a tag sequence that is substantially non-matching to the target template, in accordance with an embodiment of the invention.

[0036] FIG. 8 is a schematic diagram showing the generation of two amplicons from one ribonucleotide containing primer to incorporate a sequence that facilitates downstream amplification, according to an embodiment of the invention.

[0037] FIG. 9 is a schematic diagram showing amplification of a template using two ribonucleotide-containing primers according to an embodiment of the invention.

[0038] FIG. 10 is a schematic diagram illustrating single-tube isothermal amplification of RNA by combining ribonucleotide primer-initiated amplification with RNA polymerase, according to an embodiment of the present invention.

[0039] 11a and 11b are schematic diagrams showing an initiation and amplification cascade using ribonucleotide-containing loop primers according to an embodiment of the present invention.

[0040] FIG. 12 is a schematic diagram illustrating a strand displacement chain reaction according to an embodiment of the present invention.

[0041] 13 is a schematic diagram showing the amplification of a DNA template using a ribonucleotide-containing primer by a non-strand-displacing DNA polymerase according to an embodiment of the present invention. The amplicon has a recessive 5' end that is generated by cleavage of the ribonucleotide primer. The 3' overhang is determined by the arrangement of the ribonucleotide and the 5' sequence. If the overhangs are designed to be complementary, the amplicon forms a circular structure by annealing of the single-stranded 3' ends. The circular DNA is further transfected into a recipient cell.

[0042] Figure 14 is a schematic diagram showing target amplification and direct cloning of amplicons generated by ribonucleotide-containing primers into vectors according to an embodiment of the present invention. Amplicons generated using ribonucleotide-containing primers with a non-strand-displacing DNA polymerase are recessive at the 5' end due to RNase H2 cleavage at the 5' side of the ribonucleotide. The overhangs are designed to anneal with a preselected vector. The mixed amplicon and vector form a circular structure and can be immediately transfected into recipient cells.

[0043] FIG. 15 is a schematic diagram showing amplicon concatemerization using ribonucleotide-containing primers according to an embodiment of the present invention.

[0044] Figure 16 shows a genotyping assay using ribonucleotide-containing primers according to an embodiment of the present invention. Ribonucleotides incorporated at or near the mutation site are not cleaved by RNase H2 if they mismatch the mutation, but are cleaved if they match the target. The primers are designed with a short 3' segment, which, once cleaved and copied, will not form a stable hybridization and the primers cannot anneal in the next round of amplification. Ribonucleotides designed to match the wild-type sequence will not amplify the wild-type sequence, but will efficiently amplify any mutants.

[0045] 17 shows a haplotyping assay using ribonucleotide-containing primers according to an embodiment of the invention. Haplotypes are only efficiently amplified if both target variable sites are mismatched at or near a ribonucleotide.

[0046] Figure 18 shows the initiation of an amplification cascade using ribonucleotide-containing primers according to an embodiment of the invention. Amplification of a third target is designed to initiate only if amplification of the other two targets has occurred followed by cleavage at the nucleotide. The cleaved 5' segments of the two primers serve as primers for amplifying the third target.

[0047] FIG. 19 shows sequential initiation of amplification cascades using ribonucleotide-containing primers for multiplexed target detection according to an embodiment of the invention.

[0048] FIG. 20 shows the initiation of an amplification cascade using ribonucleotide-containing primers for target ratio imbalance detection according to an embodiment of the present invention. The 5' segments cleaved for the ribonucleotides of the first two amplicons are designed to anneal to each other in a 1:1 ratio and extend. Once the 5' segments are extended, the amplification of the third target cannot be initiated. The imbalance in the amount of starting template leads to an excess accumulation of the 5' segment of one primer, which is used to amplify the third target.

[0049] FIG. 21 is a schematic diagram showing an example of amplification of a target with an anchor primer, according to an embodiment of the present invention.

[0050] FIG. 22 is a schematic diagram showing bridge amplification using ribonucleotide-containing primers, according to an embodiment of the present invention.

[0051] FIG. 23 is a schematic diagram showing signal detection of a ribonucleotide-containing probe according to an embodiment of the present invention.

[0052] FIG. 24 shows the amplification curves of the R primers amplifying the target compared to the standard primers which show similar amplification Ct in the absence of RNase H2, but higher Ct in the presence of RNase H2.

[0053] FIG. 25 shows a comparison of melting curves of amplicons generated in the absence of R-Primer and in the presence of RNase H2.

[0054] FIG. 26 shows specific detection of EGFR exon 21 cancer mutations with R primers and a non-SD polymerase.

[0055] FIG. 27 shows the acceleration of target detection by using ribonucleotide-containing primers with RNase H2 in an isothermal LAMP assay.

[0056] FIG. 28 shows that the increase in fluorescence intensity in the isothermal reaction using the R-primer is detected earlier than that using the standard primer.

[0057] FIG. 29 shows a comparison of amplification by strand displacement chain reaction (SDCR) with standard PCR.

[0058] FIG. 30 shows the increase in fluorescence of the riboprobe detected during target amplification.

[0059] Figure 31 shows earlier detection of SARS-CoV-2 genes by real-time SDCR than PCR using standard primers.

[0060] Figure 32 shows greater than 2-fold amplification per cycle of SDCR with ribo-primers.

[0061] Terms used in the claims and specification are defined as follows, unless otherwise specified.

[0062] The term "nucleic acid" refers to a polymer of nucleotides and includes, unless otherwise limited, analogs of natural nucleotides that can function (eg, hybridize) in a manner similar to naturally occurring nucleotides.

[0063] The term nucleic acid includes any form of DNA or RNA; complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription or amplification of messenger RNA (mRNA); DNA molecules produced by synthesis or amplification; mRNA; and non-coding RNA.

[0064] The term nucleic acid encompasses not only single-stranded molecules, but also double- or triple-stranded nucleic acid complexes, in which the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).

[0065] The term nucleic acid also encompasses any modification thereof, such as by methylation and / or capping. Nucleic acid modifications include the addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interactions, and functionality into individual nucleobases or the nucleic acid as a whole. Such modifications may include base modifications such as 2' sugar modifications, 5-pyrimidine modifications, 8-purine modifications, modifications of the exocyclic amine of cytosine, substitution of 5-bromouracil, sugar-phosphate backbone modifications, and unusual base pairing. Combinations of isobases such as isocytidine and isoguanidine.

[0066] Nucleic acids can be derived from entirely chemical synthetic processes such as solid-phase mediated chemical synthesis, from biological sources such as isolation from any species that produces nucleic acids, or from processes involving the manipulation of nucleic acids using molecular biology tools such as DNA replication, PCR amplification, reverse transcription, or a combination of those processes.

[0067] The term "complementary" as used herein refers to the ability for precise pairing between two nucleotides. That is, if a nucleotide at a particular position of a nucleic acid can hydrogen bond with a nucleotide of another nucleic acid to form a standard base pair, then the two nucleic acids are considered to be complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial," where only a portion of the nucleotides bind, or complete, where complete complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

[0068] "Specific hybridization" or "specific hybridization" refers to the binding of a nucleic acid to a target nucleotide sequence under defined stringency conditions without substantial binding to other nucleotide sequences present in the hybridization mixture. Those skilled in the art will recognize this. Relaxing the stringency of hybridization conditions allows for mismatching of sequences.

[0069] In some embodiments, hybridization is performed under stringent hybridization conditions. The phrase "stringent hybridization conditions" generally refers to a temperature ranging from about 5°C. to about 20°C. or 25°C. lower than the melting temperature (Tm) of the specific sequence at a defined ionic strength and pH. As used herein, Tm is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tm of nucleic acids are well known in the art (see, e.g., Berger and Kimmel (1987) METHODS IN ENZYMOLOGY, VOL. 152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc. and Sambrook et al. (1989) Molecular Cloning: A LABORATORY MANUAL, 2nd ed., VOLS. As indicated by standard references, a simple estimate of the Tm value can be calculated by the following equation: when the nucleic acid is in an aqueous solution of 1 M NaCl, Tm = 81.5 + 0.41 (% G+C) (see, e.g., Anderson and Young, Quantitative Filter Hybridization in Nucleic Acid Hybridization (1985)). The melting temperature of a hybrid (and therefore the conditions for stringent hybridization) is influenced by a variety of factors, such as the length and nature (DNA, RNA, base composition) of the primer or probe, the nature of the target nucleic acid (DNA, RNA, base composition, present in solution or immobilized, etc.), and the concentrations of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and are discussed in standard references in the art. Exemplary stringent conditions suitable for achieving specific hybridization of most sequences are a temperature of at least about 60° C.; salt concentrations are about 0.2 molar at pH 7.Tm calculations for oligonucleotide sequences based on nearest neighbor thermodynamics are described in "A Unified View of Nearest Neighbor Thermodynamics for Polymer, Dumbbell, and Oligonucleotide DNA," John SantaLucia, Jr., PNAS, February 17, 1998, vol. 95, no. 4, 1460-1465 (hereby incorporated by reference for this discussion).

[0070] The term "oligonucleotide" refers to a relatively short nucleic acid, generally shorter than 200 nucleotides, more specifically shorter than 100 nucleotides, and most specifically shorter than 50 nucleotides. Oligonucleotides are usually single-stranded DNA molecules.

[0071] The term "DNA oligonucleotide" refers to an oligonucleotide whose components are not all DNA or RNA but are DNA analogues, e.g., DNA derivatives that contain modified bases (such as methylated bases, hydroxymethylated bases, halo-substituted bases, etc.), LNA, etc.

[0072] The term "primer" refers to an oligonucleotide that can hybridize (also called "anneal") with a nucleic acid and serve as an initiation site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., in the presence of four primers). Different nucleoside triphosphates and a polymerization agent (such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at an appropriate temperature. The appropriate length of a primer or a segment thereof depends on the intended use of the primer, but a primer is typically at least 7 nucleotides in length, and in some embodiments ranges from 10 to 30 nucleotides, or in some embodiments ranges from 10 to 60 nucleotides. Nucleotide length. In some embodiments, a primer can be, for example, 15 to 50 nucleotides in length. In general, shorter primer molecules require lower temperatures to form a sufficiently stable hybrid complex with the template. A primer does not need to reflect the exact sequence of the template, but it must be sufficiently complementary to hybridize with the template.

[0073] A primer is said to anneal to another nucleic acid when the primer or a portion thereof hybridizes to a nucleotide sequence within the nucleic acid. A statement that a primer hybridizes to a particular nucleotide sequence is not intended to mean that the primer hybridizes completely or exclusively to that nucleotide sequence. For example, in some embodiments, an amplification primer as used herein is said to "anneal" to a nucleotide sequence or to be "specific to a nucleotide sequence". This description encompasses primers that anneal entirely to a nucleotide sequence, as well as primers that anneal partially to a nucleotide sequence. Nucleotide sequence.

[0074] The term "ribonucleotide-containing primer" (also referred to herein as "riboprimer" or "R-primer") refers to a primer that contains one or more ribonucleotides. A "ribonucleotide-containing primer" anneals to a nucleic acid and initiates polymerization of nucleotides. The 3' end of the primer can have a 3'-hydroxyl group or can be blocked or activatable. The ribonucleotides in the primer can be replicated by several polymerases, particularly polymerases such as reverse transcriptase or proofreading DNA polymerases. When the primer is incorporated into double-stranded DNA, the ribonucleotides are recognized by RNase H2 and cleaved at the 5' side to form a 3' hydroxyl upstream segment and a 5' phosphate downstream segment. The ribonucleotides can be used to activate the primer when incorporated into a 3' blocked primer or to reinitiate the extension of the primer starting from the upstream segment.

[0075] The term "primer pair" refers to a set of primers that includes a 5' "upstream primer" or "forward primer" that hybridizes to the complementary strand at the 5' end of the DNA sequence to be amplified, and a 3' "downstream primer" or "reverse primer" that hybridizes to the 3' end of the sequence to be amplified. As will be appreciated by those of skill in the art, the terms "upstream" and "downstream," or "forward" and "reverse," are not intended to be limiting, but rather provide exemplary orientations in some embodiments.

[0076] A "probe" is a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation, and thus forming a duplex structure. A probe can be labeled with a detectable label to allow easy detection of the probe, especially after the probe has hybridized to its complementary target. Alternatively, however, a probe may be unlabeled but detectable by specific binding to a directly or indirectly labeled ligand. The size of a probe may vary significantly. Generally, a probe is at least 7-15 nucleotides in length. Other probes are at least 20, 30, or 40 nucleotides in length. Still other probes are somewhat longer, being at least 50, 60, 70, 80, or 90 nucleotides in length. Still other probes are even longer, being at least 100, 150, 200 or more nucleotides in length. A probe may be any length within any range bounded by any of the above values ​​(e.g., 15-20 nucleotides in length).

[0077] A primer or probe may or may not be fully complementary to a target nucleotide sequence. In some embodiments, a primer has at least 65% identity to the complement of the target nucleotide sequence over a sequence of at least 7 nucleotides, more typically over a sequence ranging from 10 to 30 nucleotides, and in some embodiments, a sequence of at least 14 to 25 nucleotides, and in some embodiments, at least 75% identity, at least 85% identity, at least 90% identity, or at least 95%, 96%, 97%, 98% identity, or 99% identity. It will be understood that it is generally desirable for certain bases (e.g., the 3' base of a primer) to be fully complementary to the corresponding base of the target nucleotide sequence. Primers and probes typically anneal to a target sequence under stringent hybridization conditions.

[0078] Amplification according to the present teachings encompasses any means by which at least a portion of at least one target nucleic acid is replicated, typically in a template-dependent manner, including, but not limited to, a wide variety of techniques for amplifying nucleic acid sequences linearly, or exponentially. Exemplary means for carrying out the amplification step include PCR, nucleic acid strand-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), loop-mediated amplification (LAMP), and the like, including multiplex versions and combinations thereof. For example, but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as multiplex chain reaction-CCR), helicase-dependent amplification (HDA), and the like. Descriptions of such techniques can be found, among other sources, in Ausubel et al.; PCR Primer: A Laboratory Manual, Diffenbach, ed., Cold Spring Harbor Press (1995); Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, edited by R. Rapley, Humana Press, Totowa, NJ (2002); Abramson et al., Curr Opin Biotechnol. 1993 Feb.;4(1):41-7, U.S. Patent No. 6,027,998; U.S. Patent No. 6,605,451, Barany et al., PCT Publication No. WO 97 / 31256.Day et al., Genomics, 29(1): 152-162 (1995); Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, 6th International Symposium on Human Identification, 1995 (available on the World Wide Web).

[0079] promega.com / geneticidproc / ussymp6proc / blegrad.html-). LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002. Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bee and Sambrook, Nucl. Acid Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Resistance 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Resistance 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18- (2002); Lage et al., Genome Res. 2003 February;13(2):294-307, and Landegren et al., Science 241:1077-80 (1988); Demidov, V., Expert Rev Mol Diagn. 2002 November;2(6):542-8., Cook et al., J Microbiol Methods. 2003 May;53(2):165-74; Schweitzer et al., Curr Opin Biotechnol. 2001 February;12(1):21-7; U.S. Patent No. 5,830,711, U.S. Patent No. 6,027,889, U.S. Patent No. 6,027,889. No. 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1.

[0080] In some embodiments, the amplification comprises annealing at least one primer to a complementary or substantially complementary sequence in at least one target nucleic acid; and using a polymerase to synthesize at least one strand of nucleotides in a template-dependent manner, and denaturing the newly formed nucleic acid duplex to separate the strands. This cycle may or may not be repeated. The amplification may include thermal cycling or may be performed isothermally.

[0081] A "multiplex amplification reaction" is a reaction in which two or more nucleic acids, distinguishable by sequence, are amplified simultaneously.

[0082] The term "qPCR" is used herein to refer to quantitative real-time polymerase chain reaction (PCR), also known as "real-time PCR" or "kinetic polymerase chain reaction." All terms refer to PCR with real-time signal detection.

[0083] "Reagent" refers broadly to any agent used in a reaction other than the analyte (e.g., the nucleic acid being analyzed). Exemplary reagents for nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases, dNTPs, etc. Reagents for enzymatic reactions include, for example, substrates, cofactors, buffers, metal ions, inhibitors, and activators.

[0084] The term "label" as used herein refers to any atom or molecule that can be used to provide detectable and / or quantifiable signals.In particular, label can be directly or indirectly attached to nucleic acid or protein.Suitable labels that can be attached to probe include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron-dense particles, magnetic particles, spin labels, chemiluminescent molecules, electrochemically active molecules, enzymes, cofactors, and enzymes.

[0085] As used herein, the term "dye" generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation.

[0086] A DNA polymerase is said to be "stable" at a particular temperature if it provides a satisfactory extension rate in a nucleic acid amplification reaction.

[0087] Figure 1 shows a scheme for using a ribonucleotide-containing primer for DNA amplification. For ease of discussion, this primer can be thought of as a "forward" primer. The ribonucleotide, when cleaved at the 5' side of the ribonucleotide, gives the primer a second opportunity to initiate replication.

[0088] In one embodiment, the primer is designed to have two segments, an upstream segment at the 5' end (Segment 1) and a downstream segment at the 3' end (Segment 2), with ribonucleotides connecting them (see Figure 1, a). The ribonucleotides also match the target template. Each segment is designed to have a high enough Tm to stably hybridize to the template under reaction conditions that allow amplification. The primer containing the ribonucleotides is first annealed to the target template (see Figure 1, b) and extended by a polymerase to create a copy of the template (i.e., the complementary strand of the template), forming a double-stranded DNA (Figure 1, c). The ribonucleotides are recognized by RNase H2 (Figure 1, d), and the enzyme cleaves at the 5' side of the ribonucleotide, generating a 3'-hydroxyl end of Segment 1 (Figure 1, e) and a 5'-phosphate ribonucleotide end of Segment 1.

[0089] The disclosed method uses RNase H to cleave the ribonucleotide-containing primer. If there is a single ribonucleotide surrounded by a DNA sequence, RNase H2 is used due to its specificity for recognizing and cleaving a single ribonucleotide. Contiguous stretches of two or more ribonucleotides can also be used within the ribonucleotide-containing primer, in which case RNase H is preferred to digest the RNA and render segment 1 extendable.

[0090] Ribonuclease H is a family of non-sequence-specific endonuclease enzymes that catalyze the cleavage of RNA in RNA / DNA substrates via a hydrolytic mechanism. RNase H2, also known as RNase H II, is a member of the RNase H family. This enzyme binds to RNA-DNA duplexes and nicks the 5' of ribonucleotides. It specifically cleaves ribonucleotides in RNA-DNA hybrids. If the sequence is single-stranded or if the ribonucleotides are mismatched, the phosphodiester bond is not hydrolyzed. Upon cleavage, the enzyme leaves a 5' phosphate end on the downstream segment and a 3' hydroxyl end on the upstream segment. RNase H2 from different sources have different enzymatic activities. RNase H2 cloned from Escherichia coli is available from New England Biolab (NEB) with optimal activity at 37°C. Thermostable RNase H2 cloned from Pyrococcus abyssi is available from Integrated DNA Technologies (IDT). It is highly active at 70-95°C and less active below 50°C. In some embodiments, E. coli RNase H2 is included in the reaction. In some embodiments, RNase H2 that is active at low temperatures is used in the reaction. When performing thermal cycling or high temperature incubations (such as PCR and isothermal amplification), it is preferable that the RNase H2 is thermostable.

[0091] In some embodiments, the polymerase has strand displacement activity (FIG. 1g). The ribonucleotide containing primer is first extended to generate a copy of the target template. The ribonucleotide is recognized and cleaved by RNase H2 enzyme to generate an extendable upstream segment 1. Segment 1 is extended from the 3'-hydroxyl group by strand displacement polymerase activity to generate a second copy of the target template, while displacing the first generated copy.

[0092] If the polymerase lacks strand displacement activity, segment 1 will not displace segment 2 and its extension product. However, segment 1 can be designed such that, once cleaved by an RNase, it is itself used as a primer.

[0093] In some embodiments, the positions of the ribonucleotides in the primer are adjusted so that Segment 1 and Segment 2 have different Tms. In a further embodiment, Segment 2 can be shortened so that it has a Tm that is too low to hybridize to the template strand by itself.

[0094] In a further embodiment, two or more ribonucleotides are included in the primer.For example, when the primer contains two ribonucleotides, the primer can further contain a third segment.After cleavage, one primer produces up to three amplicons (Figure 2).

[0095] In another embodiment, segment 1 is partially complementary to the template. For example, segment 1 has a portion complementary to the template at the 3' end. The other portions can be any of the options.

[0096] In some embodiments, the R primer includes two or more ribonucleotides. In one embodiment, the ribonucleotides are located inside the primer, separated by deoxynucleotides (Figure 2). The three segments can be separated by two ribonucleotides (Figure 2, a). The primer is extended from segment 3 by a polymerase (Figure 2, b), and one of the ribonucleotides is recognized by RNase H2, which cleaves it at its 5' side, allowing segment 2 to be extended). Extending segment 2 displaces segment 3 and the first amplicon of the template formed by the extension of segment 3. The second ribonucleotide is also recognized by RNase H2, which cleaves it at its 5' side, allowing segment 1 to be extended (Figure 2d). Extension of segment 1 by a polymerase with strand displacement activity results in the displacement of segment 2 and the second amplicon generated by the extension of segment 2. As a result, up to three amplicons are generated from one primer. In another embodiment, the ribonucleotides are located side by side within the primer (Figure 3). Each ribonucleotide, when cleaved by an RNase, can initiate extension from the 3' hydroxyl end 5' of the segment. Multiple copies of the target template can be generated from a single primer. RNase H can also be used to cleave a stretch of RNA, making segment 2 extendable to generate a second amplicon. Other ribonucleotide structures can be designed to match the specificity of the selected or engineered RNase.

[0097] RNases vary in activity and stability depending on temperature. For isothermal amplification at low temperatures, RNase H or RNase H2 can be used, which have optimal activity and stability at the target temperature. If amplification is performed at high temperatures, an RNase with high activity at the desired temperature is required. Amplification by thermocycling requires a thermostable RNase H or RNase H2 with high activity at the extension temperature.

[0098] In some embodiments, an RNA template is used with a ribonucleotide-containing primer. In Figure 4, the RNA template is reverse transcribed by a reverse transcription reaction with a ribonucleotide containing primer. The cDNA strand thus generated contains ribonucleotides in its sequence.

[0099] sample The nucleic acid template used in the present invention can be DNA or RNA, or chimeric DNA and RNA. Nucleic acid-containing samples can be obtained from biological sources and prepared using conventional methods known in the art. In particular, the nucleic acids useful in the methods described herein can be obtained from any source, including single-celled organisms and higher organisms such as plants or non-human animals, e.g., dogs, cats, horses, primates, and other non-human mammals. As well as humans. In some embodiments, the sample can be obtained from an individual suspected or known to be infected with a pathogen, an individual suspected or known to be suffering from a disease such as cancer, or an individual who is pregnant.

[0100] Nucleic acids can be obtained from cells, body fluids (e.g., blood, blood fractions, urine, etc.), or tissue samples by any of a variety of standard techniques. In some embodiments, the method uses samples of plasma, serum, cerebrospinal fluid, lymphatic fluid, peritoneal fluid, pleural fluid, oral fluid, and external sections of skin; samples from the respiratory, enterogenital, or urinary tract; tears, saliva, blood cells, stem cells, tumor samples. Samples can be obtained from live or dead microorganisms, or from in vitro cultures. Examples of samples include single cells, paraffin-embedded tissue samples, needle biopsies, etc. In some embodiments, the nucleic acid analyzed is RNA (Figure 4). RNA may be converted to DNA by reverse transcriptase, forming an RNA:DNA heteroduplex. DNA can be rendered single stranded by digestion of RNA with RNase H or the RNase H activity of reverse transcriptase.

[0101] The nucleic acid of interest can be isolated using methods well known in the art. The sample nucleic acid need not be in a pure form, but typically is sufficiently pure to enable the steps of the methods described herein to be performed.

[0102] target nucleic acid Any target nucleic acid that can be detected by nucleic acid amplification can be detected using the method described herein.In a typical embodiment, at least some nucleotide sequence information is known for target nucleic acid.For example, when the amplification reaction used is PCR, generally, sufficient sequence information is available for each end of a given target nucleic acid to allow suitable amplification primer design.

[0103] Targets may include, for example, nucleic acids associated with pathogens such as viruses, bacteria, protozoa, fungi, etc. RNA, e.g., whose over- or under-expression is indicative of disease, expressed in a tissue- or development-specific manner, or triggered by a particular stimulus; genomic DNA. Specific polymorphisms (such as SNPs), alleles, or haplotypes can be analyzed, such as in genotyping. Of particular interest is genomic DNA that is altered (e.g., amplified, deleted, and / or mutated) in genetic diseases or other pathologies; sequences associated with desirable or undesirable traits; and / or sequences that uniquely identify an individual (such as forensics or paternity testing).

[0104] In some embodiments, the target comprises the entire DNA from an organism (Figures 5 and 6). In another embodiment, the target comprises the entire cDNA from RNA. In a preferred embodiment, the entire DNA or cDNA can be amplified using ribonucleotide containing degenerate primers in the presence of RNase H2 (Figure 5a). Ribonucleotides are incorporated into the degenerate primers, and the primers can anneal to the entire target genome or transcriptome. Ribonucleotides are preferentially incorporated three bases away from the 3' end. Each annealed random primer is extended by a strand-displacing polymerase (Figure 5b). One suitable polymerase is phi29 DNA polymerase. The ribonucleotides in each extended primer are then recognized and cleaved at the 5' side of the base (Figure 5, c) and further extended from the 5' segment of the primer (Figure 5, d). Extension of the 5' segment of each primer displaces the 3' segment and the initial amplicon from the 3' end of the primer. The displaced amplicon serves as a template and anneals to a primer with a complementary sequence for further amplification. Ribonucleotide containing degenerate primers are expected to generate higher yields of whole genome amplified targets. In another embodiment, degenerate primers can be used in the presence of ribonucleotide triphosphates and RNase H2 to amplify entire DNA or cDNA (Figure 6, a). When using a polymerase that can incorporate NTPs, each annealed primer is extended by the polymerase. Ribonucleotides are randomly incorporated into the extended sequence (Figure 6, b). The newly incorporated ribonucleotides are then recognized and cleaved at the 5' side (Figure 6, c). The sequence on the 3' side of each ribonucleotide is then further extended, replacing the previous sequence in the growing amplicon (Figure 6, d). In a similar embodiment, locus-specific primers are used instead of degenerate primers in the presence of NTPs and RNase H2 to amplify target nucleic acids with a polymerase that incorporates ribonucleotides. In some embodiments, ribonucleotide-containing primers are combined with rolling circle amplification (RCA).

[0105] Primer design Suitable primers for nucleic acid amplification are long enough to initiate the synthesis of extension products in the presence of an appropriate nucleic acid polymerase. The exact length and composition of the primers depend on many factors, including the temperature of the annealing reaction, the source and composition of the primers, the location where the probe is used, the proximity of the annealing site of the probe to the annealing site of the primer, and the ratio of primer:probe concentrations. For example, depending on the complexity of the target nucleic acid sequence, oligonucleotide primers usually contain a range of about 10 to about 60 nucleotides, but can contain more or fewer nucleotides. Primers must be sufficiently complementary to selectively anneal to their respective strands and form a stable duplex.

[0106] Generally, those skilled in the art know how to design suitable primers that can amplify target nucleic acid of interest.For example, PCR primers can be designed by using any commercially available software or open source software such as Primer3 (e.g., Rozen and Skaletsky (2000) Meth. Mol. Biol., 132: 365-386; www.Broad. mit.edu / node / 1060, etc.), or access the Roche UPL website.The amplicon sequence is input into the Primer3 program together with the UPL probe sequence in brackets, and the Primer3 program designs primers on either side of the probe sequence in brackets.

[0107] Primers can be prepared by any suitable method, including direct chemical synthesis, for example, by the phosphotriester method of Narang et al. (1979) Meth. Enzyme Mol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzyme Mol. 68: 109-151; the diethyl phosphoramidite method of Beaucage et al. (1981) Tra. Lett. 22: 1859-1862; solid support methods such as U.S. Pat. No. 4,458,066 can also be utilized or provided by commercial sources. Primers can be purified by using Sephadex columns (Amersham Biosciences, Inc., Piscataway, NJ) or other methods known to those skilled in the art. Primer purification can improve the sensitivity of the methods described herein.

[0108] Ribonucleotide-containing primers (shown in FIG. 1) contain one or more ribonucleotides within a sequence and segment of a DNA oligonucleotide. They can be produced by any suitable method, including, for example, direct chemical synthesis, or can be provided from a commercial source.

[0109] "Stabilizing bases" include, for example, a series of peptide nucleic acids (PNAs) that can be incorporated into DNA oligonucleotides to increase duplex stability. Locked nucleic acids (LNAs) and unlocked nucleic acids (UNAs) are analogs of RNA that can be easily incorporated into DNA oligonucleotides during solid-phase oligonucleotide synthesis to increase and decrease duplex stability, respectively. Suitable stabilizing bases also include modified DNA bases that increase the stability of the base pair (and therefore the duplex as a whole). These modified bases can be incorporated into oligonucleotides during solid-phase synthesis, providing a more predictable method of increasing DNA duplex stability. Examples include AP-dC (G-clamp) and 2-aminoadenine, 5-methylcytosine and C(5)-propynylcytosine (replacing cytosine), and C(5)-propynyluracil (replacing thymine).

[0110] A "destabilizing base" is a base that destabilizes double-stranded DNA by forming base pairs that are less stable than typical A-T and / or G-C base pairs. Inosine (I) is a destabilizing base because it pairs with cytosine (C), but IC base pairs are less stable than GC base pairs. This less stability is due to the fact that inosine is a purine that can only form two hydrogen bonds compared to the three hydrogen bonds of a GC base pair. Other destabilizing bases will be known to or readily identified by those of skill in the art. Ribonucleotides Containing Primers in a Primer Set The ribonucleotide-containing primer can be used in the reaction together with a simple conventional reverse primer or another ribonucleotide-containing primer.

[0111] Primer with tag sequence One or more tag sequences can also be added to the 5' end of the primer (Figures 7 and 8). The 3' segment of the primer hybridizes to the target template, while the 5' segment can be partially complementary or non-complementary to the target template. The 3' segment is designed to be locus specific to the target. The tag can be composed of nucleotides that may not hybridize to the target nucleic acid. One or more ribonucleotides can be included in the locus specific region (Figure 7) or the tag region (Figure 8). The tag can be any sequence designed to facilitate downstream reactions. In one example, the tag can be a T7 promoter, M13 promoter, SP promoter, and other promoters, or combinations thereof. In some embodiments, the tag can include a nicking restriction site for a nicking enzyme assisted reaction (NEAR). In some embodiments, the tag can include a promoter and can be used to initiate in vitro transcription of the target sequence. In some embodiments, the incorporation of the tag and the reaction using the tag are included in a single tube. As shown in Figure 7, a second primer can be provided to form a primer set with a ribonucleotide-containing primer. The ribonucleotide containing primer first anneals with the template (Figure 7, a) and is extended by the polymerase (Figure 7, b). The double-stranded extension product (Figure 7, c) can then be denatured. Denaturation can be performed by increasing the temperature, alkaline denaturation or double-stranded DNA breathing. A second primer, shown in Figure 7 as a reverse primer, then anneals to the single-stranded extension product of the ribonucleotide-containing primer and makes a copy of it by the polymerase, generating the complementary strand of the tag segment (Figure 7, e). The ribonucleotide in the regenerated double-stranded amplicon is recognized by RNase H2 and cleaved at the 5' end to generate an extendable tag segment. The tag segment is then extended, displacing the sequence in front of the tag segment by the strand-displacing activity of the polymerase (Figure 7, f). As a result, the tag is incorporated into an amplifiable structure. Further amplification cycles benefit from the increased efficiency of the ribonucleotide-containing primer.

[0112] If the tag sequence in Figure 7 is designed as a functional element such as an RNA polymerase promoter or a nicking enzyme recognition site (Figure 8), the amplification of the target nucleic acid can be further combined with other nucleic acid amplification methods, such as nucleic acid sequence-based amplification (NABSA) or NEAR.

[0113] A pair of primers containing ribonucleotides In some embodiments, both the forward and reverse primers are ribonucleotide-containing primers (Figure 9). Figure 9 illustrates the schematic of the amplification process by using a pair of ribonucleotide containing primers along with a polymerase with strand displacement activity in the presence of RNase H2. The forward and reverse primers are each designed with two segments separated by a nucleotide (Figure 9, a). Primer 1, shown as the forward primer, has segment 1 (thin solid line) at its 5' end and segment 2 (thick solid line) at its 3' end. Primer 2, shown as the reverse primer, is similarly designed with two segments separated by a ribonucleotide. The 5' segment of primer 2 is shown as segment 3 (thick double line) and the 3' segment as segment 4 (thin double line). In the presence of target DNA template 1 and template 2, each primer anneals to its template and is extended by the polymerase (Figure 9, b). Extension of primer 1 from segment 2 produces amplicon 1 from template 1. Meanwhile, extension of primer 2 from segment 3 generates amplicon 2 from template 2 (Figure 9, b). Next, the ribonucleotide of primer 1 primer 2 is recognized and cleaved at the 5-side of the ribonucleotide, allowing extension of segment 1 of primer 1 and segment 3 of primer 1. Extension from segment 1 and segment 3 generates amplicons 3 and 4, respectively, but displaces the corresponding amplicons 1 and amplicons 2 generated by extension of segment 2 and segment 4 (Figure 9, c). Amplicons 3 and 4 remain mostly double-stranded and are not used for further amplification unless they are denatured as shown in Figure 10. The displaced amplicon 1 anneals with another primer 2, while the displaced amplicon 2 anneals with another primer 1 (Figure 9, c). Extension of the annealed primer 2 generates amplicon 5 from amplicon 1, and extension of the annealed primer 1 generates amplicon 6 from amplicon 2 (Figure 9, d).Recognition and cleavage of the extended primers 2 and 1 by RNase H2 renders segment 3 of primer 2 and segment 1 of primer 1 extendable, and their extension by polymerase generates amplicons 7 and 8, with displacement (Figure 9, e). Amplicon 5 and amplicon 6, respectively (Figure 9, e). The displaced amplicon 5 then anneals to segment 2 of primer 1, and amplicon 6 anneals to segment 4 of primer 2 (Figure 9, e; box 1). Extension of the 3' end of amplicon 5 makes segment 1 double-stranded, and extension of segment 2 of primer 1 from amplicon 5 generates amplicon 9 (Figure 9, f). Similarly, extension of amplicon 6 makes segment 3 double-stranded, and extension of segment 4 of primer 4 from amplicon 6 generates amplicon 10 (Figure 9, f). Next, the ribonucleotides in the double-stranded region are recognized and cleaved by RNase H2 (not shown), and then Segment 1 of Primer 1 and Segment 3 of Primer 2 are extended to generate Amplicon 11 and Amplicon 12, respectively (Figure 9, g). Meanwhile, Amplicon 9 and Amplicon 10 are transferred and annealed to Segment 4 of Primer 2 and Segment 2 of Primer 1, respectively (Figure 9, g; Box 2). The structure of Box 2 is the same as that of Box 1, and the same structure is reproduced through step f. As a result, these structures cycle continuously to generate an increased amount of amplicon in a given condition. The amplification rate increases when one or both of the primers are designed to have more segments separated by additional ribonucleotides.

[0114] Figure 10 shows one application of the amplification method shown in Figure 9. One of the ribonucleotide containing primers is designed to contain a tag. The tagged primer is used to reverse transcribe the RNA target. (Figure 10). If an RNA polymerase promoter is used as the tag, the initial incorporation of the promoter into the double-stranded DNA and the in vitro transcription (IVT) can be performed at the same temperature in the same tube. A reverse transcriptase may be further provided to amplify the target nucleic acid by nucleic acid-based amplification (NASBA). A pair of ribonucleotide containing primers is designed to amplify the target region (Figure 10, a). Either DNA or RNA can be used as the initiating template. The RNA template is shown in the figure. One of the primers (shown as a forward primer) is designed to have two segments separated by a ribonucleotide, each segment matching the target sequence. Another primer (shown as a reverse primer) is designed to one ribonucleotide in the 5' tag. The 5' tag consists of an RNA polymerase promoter that is used by the RNA polymerase to initiate the in vitro transcription (IVT). The 3' end connected to the promoter is designed to anneal to the template and initiate reverse transcription (Figure 10, a). Reverse transcriptase is used to convert the RNA to cDNA, and its RNase H activity or the natural transient dissociation of RNA from the cDNA allows annealing of the forward primer and extension of the primer (Figure 2) by either reverse transcriptase activity or an added DNA polymerase (Figure 10, b). Extension of the forward primer creates a double-stranded RNA polymerase promoter (Figure 10, c). The promoter is then used by the RNA polymerase (Figure 10, d) to transcribe the target sequence into multiple copies of RNA (Figure 10, e). The presence of ribonucleotides in the primer allows amplification of the target at the same temperature as shown in Figure 9.The newly transcribed RNA, shown in Figure 10e, further serves as a template for reverse transcription by a forward primer, and the cDNA thus generated is further amplified by a ribonucleotide-containing forward primer (Figure 10, g). The further generated double-stranded promoter triggers IVT by RNA polymerase, and the RNA is further reverse transcribed into cDNA, facilitating the next round of amplification. Cycles of reverse transcription and IVT, plus amplification by ribonucleotide containing primers, synergistically accelerate the amplification of the target nucleic acid. The use of ribonucleotide-containing primers eliminates the need for an initial thermal cycle, allowing amplification at a relatively constant temperature.

[0115] Figure 11 shows the isothermal amplification of a target using loop primers that are commonly used in LAMP assays. In this example, only two or three primers are required (Figure 11a, b). Primer B3 is optional. The forward internal primer (FIP) and backward internal primer (BIP) are each designed to have a 5' segment that forms a double-stranded stem (e.g., loop) when extended on the target template. The two primers typically follow the design of the LAMP inner primer, but with some modifications. In the FIP, ribonucleotides are incorporated near the center of the F2 and F1c regions. The Tm of each segment within F2 and F1c is high enough to form an internal loop structure at the specified reaction temperature. In the BIP, ribonucleotides are incorporated in the B1c region, and each half of the B1c segment can form an internal loop structure after cleavage of the ribonucleotide. A third optional primer, B3, can be used to displace the extension product of the BIP at the beginning (Figure 11a, b). Once the BIP extension product is separated from the template, FIP anneals to and copies the displaced BIP extension product (Fig. 11a,c). RNase H2 cleavage of FIP, extension of the F1c5' segment, and displacement of the first extension product of FIP generates the loop structure in Fig. 11a,d, which is further extended to form the long loop structure in Fig. 11,c. Cleavage and extension of the BIP primer results in two additional amplicons in Fig. 11. Each of these structures is further annealed and extended in subsequent steps (Fig. 11a,f,g) (Fig. 11,b), leading to amplification of the target.

[0116] Strand Displacement Chain Reaction (SDCR) Figure 12 shows an example of the application of the process shown in Figure 10. In this example, thermal cycling is used to denature double-stranded amplicons to initiate further rounds of amplification. If both the forward and reverse primers are ribonucleotide-containing primers, the target nucleic acid can be amplified by strand displacement chain reaction (SDCR) (Figure 12). In each circle, the template is first denatured at a temperature that dissociates the double-stranded DNA, followed by primer annealing and extension. Annealing and extension may be performed at the same temperature. As shown in Figure 10, each template is replicated multiple times, and the fold increase in each cycle depends on the annealing and extension times. When the denaturation step by thermal cycling is performed, all double-stranded amplicons are denatured and serve as templates for annealing to primers. In the annealing and / or extension steps, amplification by ribonucleotide-containing primers continues with similar efficiency as in the first cycle. If all ribonucleotide-containing primers are cleaved and extended in each cycle, the amplification efficiency is doubled compared to standard PCR. If multiple copies are generated from a single template in each cycle, the number of amplicon copies increases exponentially by more than two bases.

[0117] Polymerase The disclosed methods use a polymerase for amplification. In some embodiments, the polymerase is a DNA polymerase that lacks 5' to 3' exonuclease activity but has "strand displacement" activity. The polymerase is used under conditions such that the strand extending from the first primer can be displaced by polymerization of a second primer provided or generated by RNase H2 cleavage. Figures 2-12 show embodiments utilizing a strand displacement polymerase.

[0118] Advantageously, polymerases can displace the strand complementary to the template strand, a property called "strand displacement". Strand displacement results in the synthesis of multiple copies of the target sequence per template molecule. Exemplary polymerases with strand displacement activity include M-MuLV reverse transcriptase, phi29 DNA polymerase, DEEP VENT (exo-) DNA polymerase (all available from NEB), and SD polymerase (Bioron). If thermal cycling is performed (as in PCR), the polymerase is preferably a thermostable polymerase.

[0119] Strand displacement can also be facilitated by the use of a strand displacement factor, such as a helicase. Any DNA polymerase capable of strand displacement in the presence of a strand displacement factor is suitable for use in the disclosed methods, even if the DNA polymerase does not perform strand displacement in the absence of such a factor. Strand displacement factors useful in the methods described herein include the BMRF1 polymerase accessory subunit (Tsurumi et al., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA binding protein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164 (1994)), herpes simplex virus protein ICP8 (Boehmer and Lehman, J. Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad. Sci USA 91(22):10665-10669 (1994)), single-stranded DNA binding protein (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)), and calf thymus helicase (Sigel et al., J. Biol. Chem. 267:13629-13635 (1992)). Helicase and SSB are available in heat-stable forms, making them suitable for use in PCR.

[0120] In some embodiments, it may be advantageous to use a blend of two or more polymerases. For example, an exemplary polymerase blend includes a polymerase that is particularly skilled in initiating extension from a partially double-stranded DNA primer and a polymerase that is particularly skilled in strand displacement synthesis, since combining these properties may provide a net advantage in some embodiments. In some embodiments, reverse transcriptase is combined with DNA polymerase. DNA polymerase and reverse transcriptase may have strand displacement activity.

[0121] In some embodiments, the DNA polymerase used in the disclosed method is highly processive. Exemplary DNA polymerases include mutants of Taq DNA polymerase that lack 5' to 3' exonuclease activity, such as Taq DNA polymerase (ABI), Stoffel fragment of SD polymerase (Bioron), mutant Taq that lacks 5' to 3' exonuclease activity. US Patent No. 5,474,920, Bca polymerase (Takara), Pfx50 polymerase (Invitrogen), Tfu DNA polymerase (Qbiogene). When thermal cycling is performed (as in PCR), the DNA polymerase is preferably a thermostable DNA polymerase.

[0122] In another aspect, if it is desired to use Taqman style probes to perform real-time PCR, the polymerase blend can include a polymerase with 5' to 3' exonuclease activity that is not susceptible to "flap" endonuclease activity. In some embodiments, the probe has one or more ribonucleotides in the sequence (see example shown in Figure 23). The probe can be designed to hybridize to the amplicon and generate a large signal upon cleavage by RNase H2. In some embodiments, multiple probes can be used to hybridize to different sequences of the amplicon. The probes may be labeled with different dyes.

[0123] In some embodiments, ribonucleotide-containing primers are used with DNA polymerases that lack strand displacement activity (Figures 13, 14, 15, 16, 17, 18, 19). Ribonucleotide-containing primers can be used in PCR in the presence of thermostable RNase H2. When the ribonucleotide-containing primer is incorporated into the double-stranded amplicon, the 5'-terminal segment is cleaved. Unlike polymerases with strand displacement activity, non-strand displacing polymerases cannot extend the cleaved 5'-terminal segment. As a result, the cleaved 5'-terminal segment dissociates from the amplicon and can function as a primer. When the cleaved 5'-terminal segment dissociates, the amplicon is left with a single-stranded 3'-end.

[0124] In some embodiments, the forward and reverse primers are each designed to have a tag (Figure 13, 14) 5' of their respective locus-specific 3' ends. The locus-specific segments have an appropriate Tm to anneal to the target under specified conditions. The tags may be attached to the locus-specific sequence via a ribonucleotide at the 3' end (Figure 13, a). They may be designed to hybridize to each other such that both ends of each amplicon hybridize to each other to form a circle. During PCR amplification by thermal cycling, the ribonucleotide is recognized and cleaved at the 5' end by RNase H2 (Figure 13, b). The 5' end segment dissociates from the PCR amplicon, leaving both 3' ends single-stranded (Figure 13, c). The amplicon can anneal through intramolecular annealing of the two 3' ends, thus forming a circle (Figure 13, d). The amplicon circle can be further transfected into cells directly or after ligation. In a preferred embodiment, the circle can be treated and ligated to seal the nick. In some embodiments, the tag is designed to hybridize with the provided cloning vector with a single-stranded "sticky" end (Figure 14). The 5' ends of the two PCR primers are designed to form a sequence complementary to the vector (Figure 14, a). The target is amplified in a PCR using a non-strand displacing polymerase in the presence of RNase H2. The amplicon (Figure 14, b) will have a "sticky" end complementary to the vector upon cleavage by RNase H2 (Figure 14, c). The amplicon can be mixed with the vector for transformation or transfection of recipient cells (Figure 14, d). In a preferred embodiment, the annealed amplicon and vector can be treated and ligated to seal the nick. In some other embodiments, multiple amplicons are concatemerized by using ribonucleotide-containing primers (Figure 15). Primer pairs containing ribonucleotides are designed to amplify multiple target sequences.Each locus-specific 3' segment of the primer is designed to connect to a 5' terminal segment (15, a). The 5' terminal segment of the primer is designed to hybridize with one of the other amplicons, allowing the amplicons to join together in a predefined order when incorporated into the amplicon and digested by RNase H2 (Figure 15, b). Multiplex PCR can be used to amplify multiple target sequences. When the 5' segments are cleaved by RNase H2, the segments can anneal to form predesigned sequences.

[0125] In some embodiments, ribonucleotide-containing primers are used to differentiate or enrich for mutations in a target nucleic acid (Figure 16). A pair of primers with one ribonucleotide containing primer is used to amplify the target sequence by PCR. The ribonucleotide is designed to overlap or be located close to the target sequence variation (indicated by an X in the figure). The 3' segment of the ribonucleotide is designed to be short so that its Tm is lower than the annealing temperature. If the primer perfectly matches the target, it is cleaved by RNase H2 (Figure 16, A). The amplicon generated from the cleaved sequence does not stably anneal with the primer in the subsequent circle, resulting in near-linear amplification of perfectly matched templates. Conversely, if the primer does not match the target due to the presence of a sequence mutation, cleavage is prevented or reduced. Amplification of the mismatched template proceeds similarly to standard exponential amplification (Figure 16, b). In some further embodiments, the genotype of the mutation can be determined by a pair of ribonucleotide-containing primers. Efficient amplification indicates the presence of a sequence mutation at the ribonucleotide site. In another embodiment, variants that differ from the primer sequence are preferentially amplified and enriched. In some further embodiments, the forward and reverse primers each target one of the mutations in the target nucleic acid (Figure 17). This method is useful for determining the haplotype of the target sequence. Efficient amplification is observed when the two variations are within the same strand. The haplotype can be deduced from the amplification of the target and the identity of the ribonucleotides in the primers.

[0126] In some embodiments, ribonucleotide-containing primers can be used to initiate a cascade of reactions (see Figures 18 and 19). These methods are useful multiplex PCR alternatives for detecting multiple targets. In these designs, 5' ribonucleotide cleavage by RNase H2 in the amplicon releases the 5' end segment as a new primer for further reactions. In one example (Figure 18, A), the cleaved 5' segment of one of the primer pairs for the first target forms a primer pair with a third primer to amplify the second template. Amplification of the first template provides the primers needed to amplify the second template. Amplification of the second target is dependent on the presence and amplification of the first target. As a result, amplification and detection of the second template indicates the presence of the first and second templates. Figure 18B shows a variation of Figure 18A. In this example, two ribonucleotide containing primers are designed to amplify the first target. The 5' segments of both primers targeting the first template are used to amplify the second target. Amplification of the first target provides primers to amplify the second target. In yet another embodiment (FIG. 18C), the 5' segments from two primers from two separate primer pairs targeting the first and second templates are designed to amplify a third target. Amplification and detection of the third target is dependent on the presence and amplification of the first two templates. In another example shown in FIG. 19, the first target is amplified using a first primer pair. One primer is designed as a ribonucleotide-containing primer. The 5' terminal segment of the first ribonucleotide containing primer forms a primer pair with a third primer to amplify the second target. The third primer is designed as a ribonucleotide-containing primer whose 5' terminal segment is designed to form a primer pair with a fourth primer to amplify the third target. A cascade of amplification and release of the 5' segment as a new primer amplifies the third target. As a result, the third target is amplified only if the first and second targets are present in appropriate amounts.Amplification and detection of the third target indicates the presence of the first and second targets. In another embodiment (Figure 20), an amplification cascade is used to detect differences in target ratios. Two targets with potentially unequal ratios are amplified by two primer pairs (Figure 20, a). One primer of each pair is designed as a ribonucleotide primer. The 3' halves of the 5' end segments are designed to be complementary so that they can anneal to each other and "extinguish" each other in a 1:1 ratio. If the ratios of the first two templates are different, the amount of 5' segments produced by the amplification of the two targets will also be different. One extra amount of the 5' end segment serves as one of the primers for amplifying the third target with the fifth primer. Amplification and detection of the third template indicates that the ratios of the first and second templates are unequal. Amplification and detection of the third template indicates that the ratios of the first and second templates are unequal. This method is useful for detecting copy number variations and trisomies.

[0127] Exemplary polymerase concentrations are in the range of about 0.1-200 units per reaction. In various embodiments, the polymerase concentration is at least: 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 units per reaction or more. In some embodiments, the polymerase concentration is within a range bounded by any of these values, e.g., 0.1-1, 1-10, 10-200, 10-150, 10-100, 10-50, 20-150, 20-100, 20-50, 50-200, 50-150, 50-100, 100-200, 100-150 units per reaction, etc. If a polymerase blend is used, the total combined polymerase concentration will be any of these values ​​or fall within any of these ranges.

[0128] amplification The primer sets described above are contacted with the sample nucleic acid under conditions in which the primers anneal to their template strands (if present). The desired nucleic acid amplification method is carried out using a DNA polymerase that lacks 5'-3' exonuclease activity under the reaction conditions used. In some embodiments, a strand-displacing polymerase is advantageous. In other embodiments, a non-strand-displacing polymerase is advantageous. A reaction mixture is provided that contains appropriate components and buffer conditions to support the activity of the polymerase and RNase H. The primer sets can be conveniently added to the amplification mixture in the form of separate oligonucleotides. For example, the two primer sets can be composed of one or two ribonucleotide-containing primers. The reaction can be carried out in solution or with one or two primers immobilized on a matrix. (Figures 21, 22).

[0129] In some embodiments, the amplification step is carried out isothermally. Isothermal amplification can be carried out as strand displacement amplification, NEAR, LAMP, RT LAMP, NABSA, RPA (recombinase polymerase amplification). Primers containing RNase H2 and ribonucleotides can be used instead of all deoxynucleotide primers in certain buffers.

[0130] In some embodiments, the amplification step is carried out using PCR. To carry out a real-time PCR reaction, the reaction mixture generally contains a suitable buffer, magnesium ions (Mg) in the range of about 1 to about 10 mM, for example, in the range of about 2 to about 8 mM. 2⁺) sources, nucleotides, and, if necessary, detergents and stabilizers. An example of one suitable buffer is TRIS buffer at a concentration of about 5 mM to about 85 mM, with a concentration of 10 mM to 50 mM being preferred. In one embodiment, the concentration of TRIS buffer is 80 mM in a double strength (2x) form of the reaction mixture. The reaction mixture can have a pH range of about 7.5 to about 9.0, typically about 8.0 to about 8.5. The concentration of nucleotides can range from about 25 μM to about 1000 μM, typically about 100 μM to about 800 μM. Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700, and 800 μM. Surfactants such as Tween 20, Triton X 100, and Nonidet P40 can also be included in the reaction mixture. Stabilizers such as dithiothreitol (DTT, Cleland's Reagent) and mercaptoethanol may also be included. Additionally, the master mix may contain dUTP and uracil DNA glycosylase (uracil N-glycosylase, UNG) as needed. Master mixes are commercially available from Applied Biosystems, South San Francisco, CA (TaqMan Universal Master Mix, catalog numbers 4304437, 4318157, and 4326708).

[0131] Label Strategy Any suitable labeling strategy can be used in the methods described herein. When the reaction is analyzed for the presence of a single amplification product, a universal detection probe can be used in the amplification mixture. In certain embodiments, real-time PCR detection can be performed using a universal qPCR probe. Suitable universal qPCR probes include double-stranded DNA binding dyes such as SYBR Green, Pico Green (Molecular Probes, Inc., Eugene, Organ), Eva Green (Biotium), and ethidium bromide (see Zhu et al., 1994, Anal. Chem. 66:1941-48). In some embodiments, colorimetric dyes can be used to detect amplification. A change in pH may be used to indicate amplification. pH indicators can be used to indicate a color change when amplification causes a pH shift. Colorimetric dyes sensitive to changes in free Mg2+ concentration may be used. In some embodiments, luminescence can be used to detect amplification.

[0132] In some embodiments, one or more target-specific qPCR probes (i.e., specific for the target nucleotide sequence to be detected) are used in the amplification mixture to detect the amplification products. By judicious selection of labels, an analysis can be performed in which different labels are excited and / or detected at different wavelengths in a single reaction ("multiplex detection"). See, for example, Fluorescence Spectroscopy (Pesce et al., eds.), Marcel Dekker, New York, (1971); White et al., Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York, (1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, New York, (1971); Griffiths, Color and Structure of Organic Molecules, Academic Press, New York, (1976); Index (Bishop, ed.). Pergamon Press, Oxford, 19723; and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene (1992); and Link et al. (2017) "Multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species," PLOS One 12(5).

[0133] In some embodiments, it may be advantageous to include a label on one or more of the probes used in the amplification mixture.

[0134] Automation and Systems Examples In some embodiments, target nucleic acid is detected using an automated sample processing and / or analysis platform. In some embodiments, a commercially available automated analysis platform is utilized. For example, in some embodiments, a real-time qPCR system is utilized. However, the present invention is not limited to a specific detection method or analysis platform. Those skilled in the art will recognize that any number of platforms and methods can be utilized.

[0135] In some embodiments, the target nucleic acid is detected using a lateral flow device. A sample containing the target nucleic acid is added to the cartridge, the sample contacts a lysis buffer, and the released nucleic acid migrates along the lateral flow cartridge. The nucleic acid can then contact a reaction mixture deposited in the cartridge to initiate amplification. The amplified product can then be detected by fluorescence, luminescence, or color change.

[0136] Cartridge components include, but are not limited to, spots or lines containing lysis reagents, filters, and capture technologies that aid in the extraction, purification, and amplification of target nucleic acids. Optical windows allow for detection. kit

[0137] Also contemplated are kits for carrying out the methods described herein. Such kits include one or more reagents useful for carrying out any of these methods. The kits generally include a package with one or more containers that hold the reagents as one or more separate compositions, or optionally as a mixture where the compatibility of the reagents allows. The kits can also include other materials that may be desirable from a user's perspective, such as buffers, diluents, standards, and / or other materials useful for sample processing, washing, or carrying out other steps of the assay.

[0138] In some embodiments, the kit can include one or more riboprimers disclosed herein, a polymerase described herein, and an RNase described herein.

[0139] The kit preferably includes instructions for carrying out one or more of the amplification methods described herein. The instructions included in the kit can be affixed to the packaging material or included as a package insert. The instructions are typically written or printed, but are not limited to such. Any medium capable of storing such instructions and transmitting them to an end user can be used. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. The term "instructions" as used herein may include the address of an internet site that provides the instructions.

[0140] example Example 1. R primers amplified the target as well as standard primers, but in the presence of RNase H2, the Ct was shifted higher and shorter amplicons were generated.

[0141] Covid-19 N gene plasmid (IDT, 2019-nCoV_N_Positive Control, Cat. No. 10006625) was amplified using primers targeting the N gene sequence within the plasmid. Standard primers (Forward primer: ACT GAG GGA GCC TTG AAT ACA (SEQ ID NO:1); Reverse primer: TGC AGC ATT GTT AGC AGG AT (SEQ ID NO:2) were compared with R primer (same standard primer; SEQ ID NO:2). R-primer: ACT GAG GGA GCrC TTG AAT A+CA, +: LNA (SEQ ID NO:3)) in amplifying the target sequence. For each 10 μl reaction, 500 nM forward primer and 500 nM reverse primer were used in 1x SYBR and 30 nM ROX. For reactions with RNase H2, 2.6 mU / μl of RNase H2 (IDT, Cat. No. 11-03-02-02) was added to the reaction. Reactions were thermocycled in a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Cat. No. 4376600) and amplification was monitored in the SYBR fluorescence channel. Thermal cycling consisted of 40 cycles of 95°C / 30 s, 95°C / 15 s, and 65°C / 1 min. Amplification curves were plotted as SYBR fluorescence intensity versus cycle number in the multicomponent plot view (see Figure 24).

[0142] As shown in Figure 24, the standard all-DNA primers amplified similarly in the absence and presence of RNase H2 (right plot). The R primers performed similarly to the standard primers in the absence of RNase H2, but amplification was greatly reduced in the presence of RNase H2 (left plot).

[0143] Example 2. Short amplicons generated by R primers in the presence of RNase H2 Melting curves of the amplification products using the R primer of Example 1 were collected on a StepOnePlus Real-Time PCR System after the thermal cycling step. See Figure 25. The amplicons generated in the presence of RNase H2 have a lower Tm than the amplicons generated in the absence of RNase H2, indicating that the amplicons generated in the presence of RNase H2 are shorter and that the single ribonucleotide in the R primer is shorter than the amplicons generated in the absence of RNase H2. The double-stranded amplicons were cleaved by RNase H2.

[0144] Example 3. Specific detection of EGFR exon 21 cancer mutations using R primers and non-SD polymerase Human genomic DNA was amplified using a pair of primers, one of which was the F primer. The primers were designed to amplify a sequence in human EGFR gene exon 21. The forward R primer (CAA GAT CAC AGA TTT TGG GCrU GGC (SEQ ID NO: 4)) was designed to position a ribonucleotide base at the mutated base. The R primer was used with the reverse primer (TTT CTC TTC CGC ACC CAG (SEQ ID NO: 5)) in 1x Q5 Hot Start High Fidelity Master Mix to amplify the EFGR exon 21 sequence. Reactions consisted of 500 nM each of the forward R primer and reverse primer, 2 ng of human genomic DNA in 1x master mix, and 1x SYBR. For reactions with RNase H2, 2.6 mU / μl and 2.6 mU / μl of RNase H2 were added to each reaction. Reactions were thermocycled in a StepOnePlus real-time PCR system (Thermo Fisher Scientific) at 95°C for 2 min, 95°C for 15 s, 60°C for 30 s, and 72°C for 1 min. As shown in Figure 26, in the presence of RNase H2, amplification of the wild-type sequence was greatly reduced, whereas the mutant was effectively amplified.

[0145] Example 4. Increased signal increase by r-BIP and RNase H2 in LAMP. The LAMP assay was designed to amplify the SARS-CoV-2N gene. Two primers, B3 and BIP, each incorporated one ribonucleotide (B3: TGC AGC ATT GrUT AGC AGG+AT (SEQ ID NO: 6); BIP: AGA CGG CAT rCAT ATG GGT TGC ACG GGT GCC AAT GTG AT+CT (SEQ ID NO: 7)), while the other four, F3, FIP, LF, and LB, were standard primers (F3: TGG CTA CTA CCG AAG AGC T (SEQ ID NO: 8); FIP: TCT GGC CCA GTT CCT AGG TAG TCC AGA CGA ATT CGT GGT GG (SEQ ID NO: 9); LF: GGA CTG AGA TCT TTC ATT TTA CCG T (SEQ ID NO: 10); :1)). In the presence of RNase H2, the R primers reduced the detection time (Figure 28).

[0146] Primer concentrations in the final reaction were 200nM for F3 and B3, 1.6uM for FIP and BIP, and 400nM for LF and LB. Primers were pre-annealed with 2000 copies of Covid-19N gene plasmid (IDT, 2019-nCoV_N_Positive Control, Cat. No. 10006625) with 0.5xSYBR in a volume of 8.31μl by heating to 90°C and cooling to 20°C using a StepOnePlus Real-Time PCR System. WarmStart LAMP 2x Master Mix (NEB, E1700) was then added to the primer and plasmid mixture in each reaction well on a 96-well plate, along with 1x and 10mU / ul RNase H2 (IDT, Cat. No. 11-03-02-02). RNase H2 Dilution Buffer diluted to 0.26mU / ul was incubated at 65°C on a StepOnePlus Real Timer PCR System and SYBR fluorescence was collected at each cycle for 75 cycles (cycles / min). In this example, fluorescence was plotted against cycle number (Figure 27). The use of R primers accelerated detection of the target.

[0147] Example 5. Accelerating LAMP amplification with R primers The LAMP assay was designed to amplify the SARS-CoV-2N gene using six primers: F3: TGG CTA CTA CCG AAG AGC T (SEQ ID NO: 8); B3: TGC AGC ATT GTT AGC AGG AT (SEQ ID NO: 2); FIP: TCT GGC CCA GTT CCT AGG TAG TCC AGA CGA ATT CGT GGT GG (SEQ ID NO: 9); BIP: AGA CGG CAT CAT ATG GGT TGC ACG GGT GCC AAT GTG ATC T (SEQ ID NO: 11); LF: GGA CTG AGA TCT TTC ATT TTA CCG T (SEQ ID NO: 10): LB: ACT GAG GGA GCC TTG AAT ACA (SEQ ID NO: 1). For reactions with riboprimers, three primers, B3, BIP, and LB, each incorporated one ribonucleotide (B3: TGC AGC ATT GrUT AGC AGG +AT (SEQ ID NO:6); BIP: AGA CGG CAT rCAT ATG GGT TGC ACG GGT GCC AAT GTG AT+CT (SEQ ID NO:7); LB: ACT GAG GGA GCrC TTG AAT A+CA (SEQ ID NO:3)), while the other three, F3, FIP, and LF, incorporated a standard primer (F3: TGG CTA CTA CCG AAG AGC T (SEQ ID NO:8); FIP: TCT GGC CCA GTT CCT AGG TAG TCC AGA CGA ATT CGT GGT GG (SEQ ID NO:9); LF: GGA CTG AGA TCT TTC ATT TTA CCG T (SEQ ID NO:10)).

[0148] Primer concentrations in the final reaction were 400nM for F3 and B3, 1.6uM for FIP and BIP, and 400nM for LF and LB. Primers were pre-annealed with 2000 copies of Covid-19N gene plasmid (IDT, 2019-nCoV_N_Positive Control, Cat. No. 10006625) in a volume of 5.85μl with 1xSYBR by heating to 95°C and cooling to 20°C on a StepOnePlus Real-time PCR system. The primer and plasmid mixture in the reaction wells of a 96-well plate was then diluted with 1x WarmStart Colorimetric LAMP 2x Master Mix (NEB, M1800), 10mU / ul RNase H2 (IDT, Cat. No. 11-03-02-02; NEB, Cat. No. M0288) diluted to 0.26mU / reaction, and 3.2U / ul Bsu DNA Polymerase, Large Fragment (NEB, M0330) diluted to 2U / reaction (total volume 9.15ul) and incubated at 37°C for 30 cycles of 37°C / 15s and 37°C / 1min, 65°C / 15s and 65°C / 1min on a StepOnePlus Real Timer PCR System, collecting SYBR fluorescence at each cycle. In this example, fluorescence was plotted against cycle number (Figure 28). In the presence of RNase H2, the R primers reduced the detection time.

[0149] Example 6. SDCR R primers were used to amplify the N gene cloned into the Covid-19N gene plasmid (IDT, 2019-nCoV_N_Positive Control, Cat. No. 10006625). Each of the forward and reverse primers contained ribonucleotides in the sequence (forward r-primer: ACC AAT AGC AGT CCA GAT GAC rCAA ATT GGC TAC TAC CGA AGA GCT (SEQ ID NO: 12); reverse r-primer: GTT CCT TGA GGA AGT TGT AGC ArCG ATT GCA GCA TTG TTA GCA GGA T (SEQ ID NO: 13)). Amplification was compared to the corresponding standard primers (forward primer: TGG CTA CTA CCG AAG AGC T (SEQ ID NO: 8); reverse primer: TGC AGC ATT GTT AGC AGG AT (SEQ ID NO: 2)). 2000 copies of the N gene plasmid were amplified in 10ul reactions with 0.3U / ul of strand displacing DNA polymerase, SD polymerase (Boca Scientific, Cat. No. 1089100) using 500nM of each r-primer or standard primer. 1xSD DNA polymerase reaction buffer supplemented with 0.375mM dNTPs, 3.5mM MgCl2, 1xSYBR, and 2.6mU / ul RNase H2 (IDT, Cat. No. 11-03-02-02). Reactions were thermocycled on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Cat. No. 4376600) with 40 cycles of a 93°C / 3 min, 60°C / 1 min and 65°C / 1 min protocol, and amplification was monitored in the SYBR fluorescence channel (Figure 29). The R primers combined with a strand-displacing DNA polymerase amplified the target with a much lower Ct than the standard primers in the presence of RNase H2.

[0150] Example 7. Detection of amplification with riboprobes Real-time amplification of DNA was detected by a probe consisting of ribonucleotides without the need for 5'-nuclease activity. In a 10 μl reaction, 800 copies of the SARS-CoV-2N gene in the Covid-19N gene plasmid (IDT, 2019-nCoV_N_Positive Control, catalog number 10006625) were amplified with a pair of primers targeting the N gene (Fw primers: TCT GGC CCA GTT CCT AGG TAG TCC AGA CGA ATT CGT GGT GG (sequence number 9); Rv primers: AGA CGG CAT CAT ATG GGT TGC ACG GGT GCC AAT GTG ATC T (sequence number 11)); each at 1 nM in 1x Q5 Hot Start High-Fidelity Master Mix (NEB, catalog number M0494). Real-time amplification was performed using 5.2mU / ul RNase H2 (IDT, IDT, catalog #11-03-02-02) on a StepOnePlus Real-Time PCR System. Thermocycling conditions were 92°C / 2 min, 40 cycles of 95°C / 15 sec, 60°C / 15 sec, 70°C / 1 min. Amplification of target DNA was detected by riboprobe DNA in the presence of RNase H2 using a polymerase lacking 5'-nuclease activity (Figure 30).

[0151] Example 8. Detection of SARS-CoV-2 genes by real-time SDCR SDCR was used to amplify and detect SARS-CoV-2E genes and compared with standard primers. Forward and reverse primers were selected to target the E gene. Standard primers (Fw primer: GCT TTC GTG GTA TTC TTG CTA GTT (SEQ ID NO: 15); Rv primer: GTT AAC AAT ATT GCA GCA GTA CGC A (SEQ ID NO: 16)) contained all DNA bases, whereas ribo primers contained all DNA bases and had the same 3' and 5' halves with similar Tm separated by ribonucleotides (R-Fw primer: CGT TAA TAG TTA ATA GCG TAC TTC TTT TTC TTrG CTT TCG TGG TAT TCT TGC TAG TT (SEQ ID NO: 17)); R-Rv primer: CGT AAA AAG AAG GTT TTA CAA GAC TCA CrGT TAA CAA TAT TGC AGC AGT ACG CA (SEQ ID NO: 18)). Riboprobes were selected from the internal sequence between the forward and reverse primers, consisted of ribonucleotides and were labeled with a 5' fluorophore and a 3' quencher (R-probe: / 5HEX / ACA CTA GCC ATC rCTT ACT GCG CTT CG / 3BHQ_1 / (SEQ ID NO: 19)). To compare SDCR with standard PCR, forward and reverse were each switched with r-ribo primers or both with ribo primers. The same probe was used for all reactions. In each 10 μl reaction, 2000 copies of E gene plasmid (IDT, 2019-nCoV_E positive control, catalog number 10006896) were mixed with 500 nM primers, 250 nM probe, 3U SD polymerase (Boca Scientific, catalog number 108910) and 2.7 625 nM ROX (Lumiprobe, ROX Reference Dye for qPCR, catalog number 31110), 45 mM KCl, 4 mM MgCl2, 0.01% Triton X-100, 100 ng / ul BSA, and 0.4 mM dNTPs.Thermocycling conditions were 92°C / 2 min, 60°C / 1 min for 40 cycles. Amplification was performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) and monitored by the increase in fluorescence in the VIC channel. The logarithm of ROX-normalized HEX fluorescence intensity was plotted with cycle number. As shown in Figure 31, the R primers reduced the time required for target detection. From right to left, the amplification curves of A. standard primer, B. r-Fw + standard Rv, C. standard Fw + r-Rv primers, D. r-Fw + r-Rv primers are shown.

[0152] Example 9. SDCR Amplifies Target More Than 2-Fold Per Cycle The SDCR assay for the SARS-CoV-2E gene was compared with standard primers (Figure 32). Primer design and conditions were the same as in Example 8, except that the amount of E gene plasmid template was titrated at 40,000, 4,000, 400 and 40 copies per reaction. Ct values ​​were plotted against the logarithm of the input copy number of template. PCR efficiency was calculated as 10^(- / k), where k was the slope of the linear fit of Ct vs. log(template copy number). Standard primers amplified the target as expected by doubling every cycle, whereas SDCR amplified the target more than twice per cycle. The calculated PCR and SDCR efficiencies are shown in the table below.

[0153] [Table 1]

[0154] All publications and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0155] The steps illustrated in the drawings, described in the specification, and claimed herein are for illustrative purposes only, and it is understood that the steps do not necessarily have to be performed sequentially in the order illustrated, unless specifically required or dictated by context.

[0156] Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. Where the term "comprising" is used, it is intended to include the terms "consisting of" and "consisting essentially of." Further, the terms "comprising," "including," and "comprises" are not intended to be limiting. It is intended that the specification and examples be considered as merely exemplary, with a true scope and spirit of the invention being indicated by the claims.

Claims

1. A nucleic acid primer set comprising a first primer having a 3' end and a 5' end for amplifying a target nucleic acid in a sample, the target nucleic acid comprises a first template strand; the first primer comprises a first segment that is a DNA oligonucleotide and a second segment that is a DNA oligonucleotide; the first segment is 5' to the second segment, the first segment and the second segment are linked by at least one first ribonucleotide, and at least a portion of the second segment is capable of specifically hybridizing to the first template strand; A nucleic acid primer set comprising:

2. The primer set according to claim 1 , wherein at least one first ribonucleotide of the first primer comprises two or more ribonucleotides linked consecutively or discontinuously.

3. The primer set according to claim 1 , wherein at least one first ribonucleotide of the first primer consists of a single ribonucleotide.

4. The primer set according to claim 1 , wherein both the first segment and the second segment of the first primer specifically hybridize to the first template strand under predetermined conditions.

5. The primer set according to claim 1, characterized in that the target nucleic acid further comprises a second template strand complementary to the first template strand and a second primer capable of specifically hybridizing to the second template strand.

6. the second primer has a 3' end and a 5' end and comprises a third segment that is a DNA oligonucleotide and a fourth segment that is a DNA oligonucleotide; the third segment is 5' to the fourth segment, and the third segment and the fourth segment are linked by at least one second ribonucleotide; at least said fourth segment is capable of specifically hybridizing to a second template strand; The primer set according to claim 5 ,

7. 1. A method for amplifying a target nucleic acid comprising a first template strand in a sample, the method comprising: (a) contacting the sample with a first primer having a 3' end and a 5' end, the first primer comprising a first oligonucleotide segment and a second oligonucleotide segment, under conditions such that at least a portion of the second oligonucleotide segment anneals to the first template strand; wherein the first oligonucleotide segment is 5' to the second oligonucleotide segment and is linked to the second oligonucleotide segment by at least one first ribonucleotide; (b) extending the first primer using the first template strand as a template to generate a first amplicon complementary to the first template strand; (c) cleaving the first primer at a position 5' immediately preceding the at least one first ribonucleotide; A method for amplifying a target nucleic acid, comprising:

8. 8. The method of claim 7, wherein the cleavage step uses RNase H2.

9. said first segment anneals to said first template strand under said conditions; generating a second amplicon complementary to the first template strand by extending the first segment of the first primer using the first template strand as a template while displacing the first amplicon from the first template strand; The method according to claim 7, characterized in that

10. the first segment of the first primer does not anneal to the first template strand under said conditions; prior to the cleaving step of step (c), separating the first amplicon from the first template strand; wherein a second primer is used to generate a new nucleic acid strand complementary to said first amplicon, the new nucleic acid strand comprising a segment complementary to said first segment of said first primer; after the cleaving step (c), generating a third amplicon complementary to the new nucleic acid strand by extending the first segment of the first primer using the new nucleic acid strand as a template while displacing the first amplicon from the new nucleic acid strand; The method according to claim 7, characterized in that

11. 11. The method of claim 10, wherein at least a portion of the 3' side of the at least one first ribonucleotide and distal to the 3' end of the first primer does not specifically hybridize to the first template strand.

12. the target nucleic acid further comprises a second template strand complementary to the first template strand; (d) contacting the sample with a second primer comprising a third segment that is a DNA oligonucleotide and a fourth segment that is a DNA oligonucleotide; wherein the third segment is 5' to the fourth segment and is linked to the fourth segment by at least one second ribonucleotide, and wherein at least a portion of the fourth segment anneals to the second template strand under said conditions; (e) extending the second primer using the second template strand as a template to generate a second amplicon complementary to the second template strand; (f) cleaving the second primer at a position 5′ immediately preceding the at least one second ribonucleotide; The method according to claim 7, characterized in that

13. the first segment and the second segment both anneal to the first template strand under said conditions, and the target nucleic acid further comprises a second template strand that is complementary to the first template strand; (d) contacting the sample with a second primer comprising a third segment that is a DNA oligonucleotide and a fourth segment that is a DNA oligonucleotide; wherein the third segment is 5' to the fourth segment and is linked to the fourth segment by at least one second ribonucleotide, and both the third segment and the fourth segment anneal to the second template strand under said conditions; (e) extending a second primer using the second template strand as a template to generate a second amplicon complementary to the second template strand; (f) cleaving the second primer at a position 5' immediately preceding the at least one second ribonucleotide; (g) extending the first segment of the first primer using the first template strand as a template by displacing the first amplicon from the first template strand to generate a third amplicon that is complementary to the first template strand; extending a second primer using the second template strand as a template by displacing the second amplicon from the second template strand to generate a fourth amplicon complementary to the second template strand; The method according to claim 7, characterized in that

14. 14. The method of claim 13, further comprising the steps of: (h) generating a fifth amplicon complementary to the first amplicon using a separate second primer; and generating a sixth amplicon complementary to the second amplicon using a separate first primer.

15. increasing the temperature to separate the generated double stranded amplicons; 15. The method of claim 14, further comprising the step of decreasing the temperature to further anneal the first and second primers to the separated amplicons.

16. The method of claim 7, wherein the first primer is immobilized on a solid surface or in a matrix.

17. 8. The method of claim 7, wherein the first template strand is RNA.

18. 8. The method of claim 7, further comprising the step of (d) using the first segment cleaved from the first primer as a primer to initiate nucleotide polymerization using a second target nucleic acid.

19. 1. A method for detecting a nucleotide variation in a target nucleic acid in a sample, comprising: the sample comprises a first double-stranded DNA and a second double-stranded DNA, the second double-stranded DNA differing from the first double-stranded DNA at at least one mutation position; (a) contacting the sample with a first primer having a 3' end and a 5' end, the first primer comprising a first oligonucleotide segment and a second oligonucleotide segment; wherein the first oligonucleotide segment is 5' to the second oligonucleotide segment and is bound to the second oligonucleotide segment by at least one first ribonucleotide under conditions in which the first primer anneals to both the first template strand of the first double-stranded DNA and the first template strand of the second double-stranded DNA, but the second segment of the first primer does not anneal by itself to the first template strand of the first double-stranded DNA or the first template strand of the second double-stranded DNA; a ribonucleotide of the first primer is located at or near the mutation position, and the first primer is positioned such that it perfectly matches the first template strand of the first double-stranded DNA but does not perfectly match the first template strand of the second double-stranded DNA; (b) using the first template strand of the first double-stranded DNA as a template to generate a first amplicon by extending a first primer hybridized on the first template strand of the first double-stranded DNA, and using the first template strand of the second double-stranded DNA as a template to generate a second amplicon by extending a first primer hybridized on the first template strand of the second double-stranded DNA; (c) cleaving the first primer hybridized to the first template strand of the first double-stranded DNA at a position 5' immediately preceding the first ribonucleotide with RNase; The method is characterized in that a 5'-side segment is generated by cleaving the first primer hybridized to the first template strand of the first double-stranded DNA, while not cleaving the first primer hybridized to the first template strand of the second double-stranded DNA; (d) performing PCR to further amplify the first and second double-stranded DNAs in the presence of RNase, and distinguishing the first and second double-stranded DNAs according to the amount of each amplicon product.

20. 1. A method for amplifying a target nucleic acid in a sample comprising a first template strand, comprising: (a) contacting the sample with a first primer having a 3' end and a 5' end, the first primer comprising a first oligonucleotide segment and a second oligonucleotide segment; wherein the first oligonucleotide segment is 5' to the second oligonucleotide segment and is linked to the second oligonucleotide segment by at least one first ribonucleotide such that at least a portion of the second segment can anneal to the first template strand; (b) extending a first primer using the first template strand as a template to generate a first extension product that is complementary to the first template strand; (c) performing PCR, each amplicon comprising a first primer, to generate multiple copies of said first extension product; (d) cleaving the first primer in the amplicon of the first extension product at a position 5′ immediately preceding the at least one first ribonucleotide to generate a cleaved portion of the replicated copy; (e) annealing or ligating the cut portion of the replicated copy to another nucleic acid strand; A method for amplifying a target nucleic acid comprising the steps of:

21. comprising a quencher moiety, a fluorophore moiety, and a linker moiety connecting the quencher moiety and the fluorophore moiety; the linker moiety comprises a plurality of deoxyribonucleotides and ribonucleotides, and when the linker is intact, no fluorescence is emitted by the fluorophore; A molecular probe for detecting the amplification of a nucleic acid, comprising:

22. 22. A method for producing a method of amplification comprising the steps of: specifically hybridizing a molecular probe according to claim 21 to an amplicon product in an amplification reaction system; cleaving a ribonucleotide in the molecular probe; detecting fluorescence emitted by the fluorophore moiety of said molecular probe.

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