JP7713682B2 - Improvement of long-term nucleic acid amplification reactions - Google Patents

Description

The present invention relates to a method for performing a long-term nucleic acid amplification reaction at a specified temperature.

In recent years, various methods for performing isothermal nucleic acid amplification reactions have been developed. Methods capable of such isothermal nucleic acid amplification include, for example, the RT-RamDA method used for amplification from RNA (Patent Document 1, Non-Patent Document 1), the Multiple Displacement Amplification (MDA) method used for DNA amplification (Non-Patent Document 2), the Strand Displacement Amplification (SDA) method (Non-Patent Document 3), the Rolling Cycle Amplification (RCA) method (Non-Patent Document 4), and the Loop-Mediated Isothermal Amplification (LAMP) method (Non-Patent Document 5), which are known and have been put to practical use.

Among these, the RT-RamDA method can increase the amount of cDNA from RNA in a sample by 10 to 100 times compared to conventional reverse transcription reactions, and is a promising method that makes it possible to capture low-expressing genes and expand the number of detectable genes in gene expression analysis from trace amounts of RNA (Patent Document 1, Non-Patent Document 1). With this RT-RamDA method, it is possible to obtain a larger amount of cDNA by, for example, increasing the isothermal reaction time from 30 minutes to 120 minutes (Patent Document 1, Non-Patent Document 1). Obtaining a larger amount of cDNA makes it possible to perform various analyses from trace amounts of RNA.

International Publication No. 2016/052619

Hayashi T. , etc. al. , Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs, Nat. Commun. ,2018 Dean F. B. , etc. al. , Comprehensive human genome amplification using multiple displacement amplification, Proc. Natl. Acad. Sci. U. S. A. ,2002 Walker G. T. , etc. al. , Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system, Proc. Natl. Acad. Sci. U. S. A. , 1992 Lizardi P. M. , etc. al. , Mutation detection and single-molecule counting using isothermal rolling-circle amplification, Nat. Genet. , 1998 Notomi T. , etc. al. , Loop-mediated isothermal amplification of DNA, Nucleic Acids Res. ,2000

The present inventors have discovered, hitherto unknown, that when a nucleic acid amplification reaction is carried out over a long period of time at a specific temperature using a nucleic acid amplification reaction solution containing a hydrophilic polymer, the amplification efficiency may decrease. Therefore, the main objective of the present invention is to provide a method for carrying out a nucleic acid amplification reaction over a long period of time with high amplification efficiency even in such a case.

As a result of intensive research conducted by the present inventors to solve the above problems, the present inventors have found that in a method for amplifying nucleic acid, which includes a step of incubating a nucleic acid amplification reaction solution containing a hydrophilic polymer at a temperature higher than 30°C and lower than 50°C for a time longer than 30 minutes, the inhibition of nucleic acid amplification by the hydrophilic polymer can be suppressed by adding a specific concentration of a single-stranded DNA binding protein to the nucleic acid amplification reaction solution, and nucleic acid can be amplified with high amplification efficiency. Based on this finding, the present inventors have conducted further research and have completed the present invention.

That is, the present invention typically includes the following aspects.
[Item 1] A method for amplifying nucleic acid, comprising a step of incubating a nucleic acid amplification reaction solution containing a hydrophilic polymer at a temperature higher than 30°C and lower than 50°C for a period of time longer than 30 minutes, wherein the method suppresses inhibition of nucleic acid amplification by the hydrophilic polymer by adding 40 ng/μL or more of a single-stranded DNA binding protein to the nucleic acid amplification reaction solution.
[Item 2] The method according to item 1, wherein the nucleic acid is amplified by the RT-RamDA method.
[Item 3] The method according to Item 1 or 2, wherein the nucleic acid amplification reaction solution contains a DNA strand-specific RNA:DNA hybrid strand degrading enzyme, 40 ng/μL or more of a single-stranded DNA binding protein, a reverse transcriptase, a primer, a template RNA, and a hydrophilic polymer.
[Item 4] The method according to Item 3, wherein the DNA strand-specific RNA:DNA hybrid strand degrading enzyme is a non-specific DNA degrading enzyme.
[Item 5] The method according to Item 3 or Item 4, wherein the template RNA is RNA extracted from 1 cell to 1000 cells.
[Item 6] The method according to any one of Items 1 to 5, wherein the single-stranded DNA binding protein is T4 gene 32 protein.
[Item 7] The method according to any one of Items 1 to 6, wherein the incubation time is 60 minutes or longer.
[Item 8] The method according to any one of Items 1 to 7, wherein the incubation time is 120 minutes or longer.
[Item 9] The method according to any one of Items 1 to 8, wherein the hydrophilic polymer is at least one selected from the group consisting of a nucleic acid polymer and polyethylene glycol.
[Item 10] The method according to any one of Items 1 to 9, wherein the concentration of the hydrophilic polymer in the nucleic acid amplification reaction solution is 0.01 ng/μL or more.
[Item 11] The method according to any one of Items 1 to 10, wherein the hydrophilic polymer is at least one selected from the group consisting of polyinosinic acid and polycytidylic acid.
[Item 12] The method according to any one of Items 1 to 11, wherein the incubation temperature is 35° C. or higher and 40° C. or lower.
[Item 13] A method for amplifying nucleic acid, comprising a step of incubating a nucleic acid amplification reaction solution containing a hydrophilic polymer at a temperature higher than 30°C and lower than 50°C for a time longer than 30 minutes, wherein the nucleic acid amplification reaction solution contains 0.01 to 1,500 parts by mass of a single-stranded DNA binding protein per part by mass of the hydrophilic polymer, thereby suppressing inhibition of nucleic acid amplification by the hydrophilic polymer.
[Item 14] A kit used for nucleic acid amplification, which includes a step of incubating a nucleic acid amplification reaction solution at a temperature higher than 30°C and lower than 50°C for a period of time longer than 30 minutes, the kit comprising a hydrophilic polymer and a single-stranded DNA binding protein at a concentration adjusted to be 40 ng/μL or more in the nucleic acid amplification reaction solution.
[Item 15] A kit used for nucleic acid amplification, which includes a step of incubating a nucleic acid amplification reaction solution at a temperature higher than 30°C and lower than 50°C for a period of time longer than 30 minutes, the kit comprising a hydrophilic polymer and 0.01 to 1,500 parts by mass of a single-stranded DNA binding protein per 1 part by mass of the hydrophilic polymer.

The present invention allows nucleic acid amplification reactions to be carried out for long periods of time, for example, even in the presence of hydrophilic polymers, without impairing amplification efficiency. In particular, even when amplifying cDNA from template RNA using the RT-RamDA method or the like, it is possible to suppress inhibition of amplification caused by extending the reaction time, and to carry out long-term reactions with high amplification efficiency. This makes it possible to carry out nucleic acid amplification methods such as the RT-RamDA method under a variety of conditions, which is useful for the advancement of genetic analysis technology. For example, it will contribute to the promotion of research into single-cell analysis using the RT-RamDA method, which is applied to technologies such as next-generation sequencers (NGS) and real-time polymerase chain reaction (PCR).

1 shows the results of real-time PCR in Example 1. The amplification rate when the RT-RamDA reaction was carried out at 37° C. for 120 minutes was compared depending on the concentration of polyinosinic acid added. The amplification rate was evaluated for the βACTIN gene, and the vertical axis represents the difference from the normal reverse transcription reaction without the RT-RamDA reaction as the ΔCt value. 2 is a diagram showing the results of real-time PCR in Example 2. The difference in amplification rate depending on the amount of reverse transcriptase or single-stranded DNA binding protein added was evaluated when polyinosinic acid was added and the RT-RamDA reaction was carried out at 37° C. for 30, 120, and 360 minutes. The βACTIN gene and the NEAT1 gene were evaluated, and the vertical axis was used to evaluate the amplification rate, with the difference from a normal reverse transcription reaction without the RT-RamDA reaction being the ΔCt value. 3 is a diagram showing the results of real-time PCR in Example 3. The same evaluation as in Example 2 (FIG. 2) was performed using different types of reverse transcriptase. The evaluation was performed for the βACTIN gene and the NEAT1 gene, and the amplification rate was evaluated by taking the difference from a normal reverse transcription reaction without the RT-RamDA reaction as the ΔCt value on the vertical axis. 4 is a diagram showing the results of real-time PCR in Example 4. The concentration dependency of the inhibitory effect of single-stranded DNA binding protein on the RT-RamDA reaction of polyinosinic acid was evaluated. The βACTIN gene was evaluated, and the amplification rate was evaluated by taking the difference from the normal reverse transcription reaction without the RT-RamDA reaction as the ΔCt value on the vertical axis. 5 shows the results of real-time PCR in Example 5. The inhibitory action of polyinosinic acid on the RT-RamDA reaction and its effect on the reaction were also confirmed for polycytidylic acid, a different nucleic acid polymer. The βACTIN gene and NEAT1 gene were evaluated, and the amplification rate was evaluated by taking the difference from the normal reverse transcription reaction without the RT-RamDA reaction as the ΔCt value on the vertical axis. 6 shows the results of real-time PCR in Example 6. The inhibitory action of polyinosinic acid on the RT-RamDA reaction and its effect on the reaction were also confirmed with polyethylene glycol, a different hydrophilic polymer. The βACTIN gene was evaluated, and the amplification rate was evaluated by taking the difference from the normal reverse transcription reaction without the RT-RamDA reaction as the ΔCt value on the vertical axis.

The present invention will be described in further detail below with reference to embodiments of the present invention.
<Method for suppressing inhibition of nucleic acid amplification by hydrophilic polymer in a method for amplifying nucleic acid>
In one embodiment, the present invention provides a method for amplifying nucleic acid, comprising a step of incubating a nucleic acid amplification reaction solution containing a hydrophilic polymer at a temperature higher than 30° C. and lower than 50° C. for a time longer than 30 minutes, in which inhibition of nucleic acid amplification by the hydrophilic polymer is suppressed by including a single-stranded DNA binding protein in the nucleic acid amplification reaction solution at a specific concentration (or at a specific quantitative ratio to the hydrophilic polymer). The method can be used in any nucleic acid amplification that includes at least a step of incubation under the above conditions. Preferably, the method can be used when amplifying nucleic acid by the RT-RamDA method, but is not particularly limited thereto.

1. Nucleic Acid Amplification Reaction Solution The nucleic acid amplification reaction solution contains components generally used in nucleic acid amplification in addition to a hydrophilic polymer and a single-stranded DNA binding protein.
In one embodiment, the nucleic acid amplification reaction solution preferably contains a template nucleic acid, a primer, deoxyribonucleotides, a DNA polymerase, a hydrophilic polymer, and a single-stranded DNA binding protein, and may optionally contain other components such as a reverse transcriptase.
In another embodiment, the nucleic acid amplification reaction solution preferably contains the components used in the RT-RamDa method, a hydrophilic polymer, and a single-stranded DNA binding protein. The RT-RamDA method is a method for amplifying nucleic acids, which generally includes a step of incubating a mixture containing a template RNA, a primer, a DNA strand-specific RNA:DNA hybrid strand degrading enzyme, an RNase H minus type reverse transcriptase, and a substrate. In the RT-RamDA method, a complementary strand DNA (cDNA) of the template RNA is synthesized by the RNA-dependent DNA polymerase activity of the RNase H minus type reverse transcriptase, the cDNA strand of the hybrid strand of RNA and cDNA is randomly cleaved by the DNA strand-specific RNA:DNA hybrid strand degrading enzyme, and the cleavage site serves as a starting point, whereby the 3' side cDNA strand is peeled off from the RNA by the strand displacement activity of the RNase H minus type reverse transcriptase, and a new cDNA strand is synthesized at the part peeled off by the RNase H minus type reverse transcriptase. Details of the RT-RamDA method are described in U.S. Patent Application Publication No. 2017/0275685 (the entirety of which is incorporated herein by reference), etc. Therefore, when amplifying a nucleic acid by the RT-RamDA method, the nucleic acid amplification reaction solution contains, for example, a template RNA, a primer, deoxyribonucleotides, a reverse transcriptase, a DNA strand-specific RNA:DNA hybrid strand degrading enzyme, a hydrophilic polymer, and a single-stranded DNA binding protein. In addition, the nucleic acid amplification reaction solution may also contain other components such as a DNA polymerase.

(1) Hydrophilic polymers Hydrophilic polymers are added for various purposes, such as to increase the accuracy and synthesis rate when synthesizing and amplifying cDNA from template RNA in nucleic acid amplification methods such as the RT-RamDA method, and to inhibit cDNA synthesis from specific RNAs (e.g., rRNAs that are not intended to be synthesized). However, it has become clear from the results of the test examples described below that the nucleic acid amplification reaction is inhibited when a nucleic acid amplification reaction solution containing a hydrophilic polymer is incubated at a predetermined temperature for a long period of time. Without being bound by a specific theory, it is believed that this is because the hydrophilic polymer binds to enzymes and components necessary for nucleic acid amplification reactions (e.g., RT-RamDA reactions), such as reverse transcriptase and single-stranded DNA binding protein, and reduces the activity of these enzymes and components. Therefore, the hydrophilic polymer can be an inhibitor that reduces the amplification efficiency in nucleic acid amplification, which includes, for example, a process of incubating a nucleic acid amplification reaction solution at a predetermined temperature for a long period of time. Examples of such hydrophilic polymers include, but are not limited to, nucleic acid polymers, heparin, polyethylene glycol, and the like. These may be used alone or in combination of two or more types. Among them, nucleic acid polymers and/or polyethylene glycol are highly likely to cause inhibition, and among nucleic acid polymers, polyinosinic acid and/or polycytidylic acid are particularly likely to cause inhibition.
The nucleic acid amplification conditions (e.g., conditions of the RT-RamDA method) under which the amplification efficiency is reduced by the hydrophilic polymer are not particularly limited as long as the conditions are conditions for incubation at a temperature higher than 30°C and lower than 50°C for a time longer than 30 minutes, and may be isothermal conditions or thermal cycle conditions. In particular, inhibition tends to occur easily when the incubation time is longer than 30 minutes at a temperature of 35°C to 45°C, especially at a temperature of 35°C to 40°C. In particular, inhibition is strong at an incubation time of 60 minutes or more at the temperature, and even stronger inhibition occurs at an incubation time of 120 minutes or more at the temperature. In addition, even stronger inhibition may occur at an incubation time of 240 minutes or more at the temperature, and even stronger inhibition may occur at an incubation time of 360 minutes or more at the temperature. The upper limit of the incubation time at the temperature is not particularly limited, but may be, for example, 420 minutes or less or 360 minutes or less.
The lower limit of the concentration of the hydrophilic polymer in the nucleic acid amplification reaction solution is not particularly limited as long as the effect of the present invention can be exhibited, but for example, it is 0.01 ng/μL or more, 0.1 ng/μL or more, or 1 ng/μL or more relative to the entire nucleic acid amplification reaction solution. The upper limit of the concentration of the hydrophilic polymer in the nucleic acid amplification reaction solution is also not limited, but for example, it can be 3 μg/μL or less. In addition, when the hydrophilic polymer is a nucleic acid polymer, it can be, for example, 2 ng/μL or less.
For example, when a nucleic acid amplification reaction is performed together with a reverse transcription reaction, the addition of a nucleic acid polymer to a nucleic acid amplification reaction solution is useful for suppressing reverse transcription of ribosomal RNA (International Publication No. 2020/184551 (the entirety of which is incorporated herein by reference)). Examples of nucleic acid polymers include inosinic acid polymers, cytidylic acid polymers, guanylic acid polymers, adenylic acid polymers, thymidylic acid polymers, uridylic acid polymers, deoxyinosinic acid polymers, deoxycytidylic acid polymers, deoxyguanylic acid polymers, deoxyadenylic acid polymers, deoxythymidylic acid polymers, and deoxyuridylic acid polymers. Here, for example, the term "inosinic acid polymer" refers to an inosinic acid homopolymer (polyinosinic acid), an inosinic acid copolymer (e.g., a copolymer in which the constituent units derived from inosinic acid are more than 50 mol%, 60 mol% or more, 70 mol% or more, 80 mol% or more, or 90 mol% or more, but less than 100 mol%), derivatives thereof (e.g., those in which functional groups such as fluorine atoms, bromine atoms, iodine atoms, alkyl groups, amino groups, mercapto groups, etc. are introduced into at least a portion of the base moiety, and/or those in which at least a portion of the phosphoric acid moiety is replaced with a thiophosphoric acid moiety), and salts thereof (e.g., alkali metal salts such as sodium salts and potassium salts), and also refers to a double-stranded polymer called poly(I:C) in which polyinosinic acid and polycytidylic acid are annealed. The term is used in the same sense for other cytidylic acid polymers, etc.
Examples of nucleic acid polymers that particularly reduce amplification efficiency include at least one selected from the group consisting of inosinic acid polymers, cytidylic acid polymers, guanylic acid polymers, deoxyinosinic acid polymers, deoxycytidylic acid polymers, and deoxyguanylic acid polymers, and inosinic acid polymers and/or deoxyinosinic acid polymers, cytidylic acid polymers and/or deoxycytidylic acid polymers particularly reduce amplification efficiency. The nucleic acid polymer may be of any length, and may be, for example, a nucleic acid polymer having a total length of 30 to 10,000 bases.

(2) Single-stranded DNA binding protein Examples of single-stranded DNA binding proteins include T4 gene 32 protein, RecA, SSB (Single-Stranded DNA Binding Protein), and combinations of two or more of these. In the present invention, by setting the concentration of the single-stranded DNA binding protein to 40 ng/μL or more, the amplification efficiency can be increased in nucleic acid amplification including a step of incubating at a temperature higher than 30° C. and lower than 50° C. for a period longer than 30 minutes. Without being bound by any particular theory, this is believed to be because increasing the concentration of the single-stranded DNA binding protein causes it to bind to a hydrophilic polymer and suppress the inhibitory effect of the hydrophilic polymer on the nucleic acid amplification reaction (for example, the RT-RamDA reaction).
The concentration of the single-stranded DNA binding protein in the nucleic acid amplification reaction solution is not particularly limited as long as it is 40 ng/μL or more, but is preferably 45 ng/μL or more, more preferably 50 ng/μL or more, even more preferably 55 ng/μL or more, even more preferably 60 ng/μL or more, and particularly preferably 65 ng/μL or more. The upper limit of the concentration of the single-stranded DNA binding protein in the nucleic acid amplification reaction solution is not particularly limited, but can be, for example, 150 ng/μL or less, 140 ng/μL or less, 130 ng/μL or less, 120 ng/μL or less, 110 ng/μL or less, or 100 ng/μL or less. By adding the single-stranded DNA binding protein to the nucleic acid amplification reaction solution at such a concentration, even in nucleic acid amplification that includes a step of incubating a nucleic acid amplification reaction solution containing a hydrophilic polymer at a predetermined temperature for a long period of time, the inhibition of nucleic acid amplification by the hydrophilic polymer is suppressed, and the amplification efficiency can be sufficiently maintained.
In one embodiment, the content of the single-stranded DNA binding protein is preferably 0.01 parts by mass or more, more preferably 0.015 parts by mass or more, and more preferably 0.02 parts by mass or more, relative to 1 part by mass of the hydrophilic polymer. In a specific embodiment, when the hydrophilic polymer is a nucleic acid polymer, the content of the single-stranded DNA binding protein is preferably 10 parts by mass or more (or more than 10 parts by mass) relative to 1 part by mass of the nucleic acid polymer, more preferably 15 parts by mass or more, even more preferably 20 parts by mass or more, even more preferably 30 parts by mass or more, and particularly preferably 40 parts by mass or more. In addition, the content of the single-stranded DNA binding protein is preferably 1500 parts by mass or less, more preferably 1000 parts by mass or less, even more preferably 500 parts by mass or less, and particularly preferably 100 parts by mass or less, relative to 1 part by mass of the hydrophilic polymer. In a specific embodiment, when the hydrophilic polymer is polyethylene glycol, the content of the single-stranded DNA binding protein is preferably less than 10 parts by mass, more preferably 5 parts by mass or less, even more preferably 1 part by mass or less, even more preferably 0.5 parts by mass or less, and particularly preferably 0.1 parts by mass or less, relative to 1 part by mass of polyethylene glycol. By allowing the hydrophilic polymer and the single-stranded DNA binding protein to coexist at such a ratio, the effects of the present invention can be more reliably exhibited.

(3) DNA strand-specific RNA:DNA hybrid strand degrading enzyme The DNA strand-specific RNA:DNA hybrid strand degrading enzyme is preferably an enzyme having an activity of cleaving a DNA strand in a hybrid strand of RNA and DNA. As the enzyme, for example, a double-strand-specific DNA degrading enzyme or a non-specific DNA degrading enzyme can be used. Among these, the non-specific DNA degrading enzyme is preferred in the present invention.

The double-strand-specific DNA enzyme (also referred to as double-strand-specific nuclease; DSN) may be an enzyme derived from a prokaryote or a eukaryote, but preferably, a double-strand-specific DNA enzyme derived from a crustacean or a modified form thereof may be used. Specific examples include the following.
・ Solenocera melanthho (shrimp shrimp) DNase
・ Penaeus japonicus (prawn) DNase
・ Paralithodes camtschaticus (red king crab) DSN
・ Pandalus borealis (northern red shrimp) dsDNase
・ Chionoecetes opilio (snow crab) DSN
Other DSN Homologs The double-strand-specific DNA enzyme is preferably an enzyme that has DNA degradation activity even at temperatures below 60° C. Among the above, shrimp-derived double-strand-specific DNA enzyme or a modified form thereof is preferred.
Commercially available double-stranded specific DNA decomposition enzymes can be used, including dsDNase (ArcticZymes), Hl-dsDNase (ArcticZymes), dsDNase (Thermo scientific), Shrimp DNase, Recombinant (Affymetrix), Atlantis dsDNase (Zymo Research), and Thermolabile Nuclease (Roche).

The non-specific DNA degrading enzyme may be an enzyme that has an activity of cleaving the DNA strand of an RNA-DNA hybrid chain, but does not substantially have an activity of cleaving the RNA strand of an RNA-DNA hybrid chain or single-stranded RNA, and preferably has an activity of cleaving single-stranded DNA lower than that of cleaving the DNA strand of an RNA-DNA hybrid chain. The non-specific DNA degrading enzyme is preferably an enzyme that has DNA degrading activity even at temperatures below 60° C. As such a non-specific DNA degrading enzyme, a commercially available product may be used, for example, DNase I (manufactured by Thermo Fisher, DNase I) or the like.
The non-specific DNA enzyme to be used may be an enzyme derived from a prokaryote or a eukaryote. Preferably, a non-specific DNA enzyme derived from a mammal or a modified version thereof, more preferably a non-specific DNA enzyme derived from a bovine or a modified version thereof, may be used.

The above-mentioned modified form refers to an enzyme obtained by modifying a naturally occurring amino acid sequence. Specifically, it is an enzyme consisting of an amino acid sequence that has 80% or more (preferably 90% or more, more preferably 95% or more) sequence identity with a naturally occurring amino acid sequence, as well as an enzyme consisting of an amino acid sequence in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) amino acids have been deleted, substituted, and/or added in the naturally occurring amino acid sequence.

(4) Primer The primer may be a specific primer for a template nucleic acid such as a template RNA, an oligo dT primer, a random primer, or a combination of two or more of these. Among these, an oligo dT primer or a random primer, unlike a primer specific to a specific sequence, can anneal to any RNA to initiate reverse transcription of a wide variety of RNAs, and is therefore preferred in situations where DNA synthesis by comprehensive reverse transcription of RNA is required. In certain embodiments, it is particularly useful to carry out the reverse transcription reaction using a combination of an oligo dT primer and a random primer. The molar ratio of the oligo dT primer to the random primer is, for example, 1:5 to 1:15, preferably 1:8 to 1:12.
Examples of random primers include completely random primers and NSR (Not So Random) primers.
A completely random primer is a mixture of primers having various base sequences, each of which is a completely random base sequence. A completely random primer may include a sequence that is completely identical (or completely complementary) to an rRNA sequence. Examples of completely random primers include completely random pentamers, completely random hexamers, completely random heptamers, completely random octamers, and combinations thereof. For example, a completely random hexamer may be a mixture of all possible base sequences ( ⁴⁶ types) with four types of nucleotides (A, T, C, G).
The NSR primer is a complete random primer from which a primer having a sequence completely complementary to an rRNA sequence has been removed. Examples of the rRNA sequence to be removed include 18S rRNA sequences, 28S rRNA sequences, 12S rRNA sequences, 16S rRNA sequences, and combinations thereof.
The NSR primer may be, for example, a completely random hexamer excluding hexamers having sequences completely complementary to rRNA sequences. Also, a primer set such as a completely random pentamer, a completely random heptamer, or a completely random octamer excluding those having sequences completely complementary to rRNA sequences may be used as the NSR primer.
By using such an NSR primer, it is possible to suppress the transcription of rRNA, which is said to account for approximately 80 to 90% of RNA in the body, and therefore it is possible to relatively increase the amount of transcription of RNA other than rRNA, thereby improving the sensitivity, for example, in analyzing mRNA, etc.
For details of NSR primers, see 1) Amour et al., Digital transcriptome profiling using selective hexamer priming for cDNA synthesis, Nature Methods, Vol. 6, No. 9, 2009, pp. 647-649, 2) Ozsolak et al., Digital transcriptome profiling from attomole-level RNA samples, Genome Research, Vol. 20, 2010, pp. 519-525, 3) U.S. Patent Application Publication No. 2010/0029511, the entirety of which is incorporated herein by reference.
The length of the primer is, for example, 5 bases or more, preferably 6 bases or more, from the viewpoint of annealing, and, for example, 30 bases or less, preferably 25 bases or less, more preferably 20 bases or less, from the viewpoint of synthesis.
In the nucleic acid amplification reaction solution, the concentration of the primer is not particularly limited, but is, for example, 1 to 10 μM, preferably 2 to 6 μM, and more preferably 3 to 5 μM.

(5) Deoxyribonucleotide As deoxyribonucleotide, deoxyribonucleoside triphosphate is preferred.As deoxyribonucleotide triphosphate, for example, deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyuridine triphosphate (dUTP), their derivatives, and combinations of two or more of them can be mentioned.Among these, the mixture of dCTP, dGTP, dATP, and dTTP, the mixture of dCTP, dGTP, dATP, and dUTP, the mixture of dCTP, dGTP, dATP, dTTP, and dUTP, etc. are preferred.

(6) Reverse Transcriptase Reverse transcriptase refers to any protein (enzyme) having reverse transcription activity (RNA-dependent DNA polymerase activity), and is not particularly limited, but a polymerase exhibiting reverse transcriptase activity is preferred. In addition, the reverse transcriptase is preferably one having low RNase H activity or no RNase H activity. Examples of reverse transcriptase include avian myeloblastosis virus reverse transcriptase (AMV-RT), Moloney Murine Leukemia Virus reverse transcriptase (MMLV-RT), human immunovirus reverse transcriptase (HIV-RT), EIRV-RT, RAV2-RT, C. Examples of the polymerase include C. hydrogenogormans DNA polymerase, rTth DNA polymerase, SuperScript I, SuperScript II, mutants thereof, and derivatives thereof. Among these, MMLV-RT is preferred.

(7) DNA polymerase The nucleic acid amplification reaction solution may or may not contain the following DNA polymerase:
Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT™, DEEPVENT™, and variants thereof

(8) RNase Inhibitor The nucleic acid amplification reaction solution may contain an RNase inhibitor. The RNase inhibitor is not particularly limited, and examples thereof include proteins derived from human placenta, rat lung, or pig liver.

(9) Template Nucleic Acid In the present invention, the template nucleic acid to be used is not particularly limited, but is preferably RNA. The template RNA is not particularly limited, but is preferably RNA extracted from 1 cell to 1000 cells, and more preferably RNA extracted from 1 cell to 100 cells. Particularly preferably, RNA is extracted from 1 cell. The number of cells can be appropriately adjusted using a cell sorter. Extraction of RNA from cells can be performed by any RNA extraction method known in the art, and may be performed using a commercially available RNA extraction kit or the like.

(10) Additives The nucleic acid amplification reaction solution may further contain other additives, such as a buffer, a salt, a surfactant, or a combination of two or more of these.
Examples of the buffer include Tris, Bis-Tris, Tricine, Bis-Tricine, Hepes, Mops, Tes, Taps, Pipes, Caps, and combinations of two or more of these. The buffer is usually dissolved in water (preferably nuclease-free water) and used in the form of an aqueous solution.
Examples of salts include chlorides (e.g., lithium chloride, sodium chloride, potassium chloride, magnesium chloride, manganese chloride), acetates (e.g., lithium acetate, sodium acetate, potassium acetate, magnesium acetate, manganese acetate), sulfates (e.g., potassium sulfate, magnesium sulfate, manganese sulfate), and combinations of two or more of these.
Examples of the surfactant include anionic surfactants (e.g., sodium dodecyl sulfate, sodium cholate, sodium deoxycholate), cationic surfactants (e.g., cetyltrimethylammonium bromide), nonionic surfactants (e.g., octylphenol ethoxylate, polyoxyethylene lauryl ether, polyoxyethylene oleyl ether, polyoxyethylene stearyl ether, polyoxyethylene sorbitan monolaurate), and amphoteric surfactants (e.g., 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). These may be used alone or in combination of two or more. In a particularly preferred embodiment, a nonionic surfactant can be used.

2. Incubation The method for amplifying nucleic acid includes at least a step of incubating at a temperature higher than 30° C. and lower than 50° C. for a time longer than 30 minutes. When incubation under such conditions is performed with a nucleic acid amplification reaction solution containing a hydrophilic polymer, amplification inhibition may occur over time, but according to the present invention, the amplification inhibition can be effectively suppressed. The step of incubating at a temperature higher than 30° C. and lower than 50° C. for a time longer than 30 minutes may be under isothermal conditions or thermal cycle conditions. When the step of incubating at a temperature higher than 30° C. and lower than 50° C. for a time longer than 30 minutes is performed under thermal cycle conditions, the total time of all the incubation times under two or more temperature conditions within this temperature range is longer than 30 minutes.

In one embodiment, when nucleic acid amplification from template RNA is performed by the RT-RamDA method, the nucleic acid amplification reaction solution may be incubated under isothermal conditions or thermal cycle conditions, with isothermal conditions being preferred.

(1) Isothermal Conditions When incubation (particularly, incubation in the RT-RamDA method) is performed under isothermal conditions, it can be performed at a predetermined temperature between 30° C. and 50° C., preferably between 35° C. and 45° C., more preferably between 35° C. and 40° C., for example, 37° C., and can be performed at that temperature for a predetermined time (for example, more than 30 minutes and not more than 420 minutes, preferably 60 to 420 minutes, more preferably 120 to 420 minutes).
The incubation at a predetermined temperature between 30° C. and 50° C. may be performed in two or more stages. For example, the incubation may be performed at a predetermined temperature between 30° C. and 35° C. for 5 to 15 minutes, and then at a predetermined temperature between 35° C. and 50° C. for a predetermined time (e.g., 20 to 60 minutes).
After incubation for more than 30 minutes at a predetermined temperature between higher than 30° C. and lower than 50° C., incubation may be performed at a predetermined temperature between, for example, 50° C. or higher and lower than 100° C. Incubation at a predetermined temperature between 50° C. or higher and lower than 100° C. may be performed in two or more stages. For example, incubation may be performed at a predetermined temperature between 50° C. or higher and lower than 80° C. for 5 to 15 minutes, and then at a predetermined temperature between 80° C. or higher and 90° C. for 5 to 15 minutes.

(2) Thermal cycle conditions When incubation (particularly incubation in the RT-RamDA method) is performed under thermal cycle conditions, a predetermined temperature T1 (e.g., 33°C) between 30°C and less than 35°C and a predetermined temperature T2 (e.g., 37°C) between 35°C and 45°C are combined, and a predetermined time at T1 (e.g., 1 to 3 minutes, for example, 2 minutes) and a predetermined time at T2 (e.g., 1 to 3 minutes, for example, 2 minutes) are set as one cycle, and this may be repeated preferably for 10 to 40 cycles, more preferably for 15 to 35 cycles, to react for a time longer than 30 minutes. In addition, prior to the above thermal cycle, for example, incubation may be performed for a predetermined time (e.g., 5 to 15 minutes) at a predetermined temperature between 25°C and less than 30°C, then for a predetermined time (e.g., 5 to 15 minutes) at a predetermined temperature between 30°C and less than 35°C, and then for a predetermined time (e.g., 1 to 5 minutes) at a predetermined temperature between 35°C and less than 50°C. After the above-mentioned thermal cycle, the mixture may be incubated at a predetermined temperature between 50° C. and less than 80° C. for a predetermined time (e.g., 5 to 15 minutes), and then at a predetermined temperature between 80° C. and 90° C. for a predetermined time (e.g., 5 to 15 minutes).

<Nucleic acid amplification kit>
As described above, according to the present invention, a nucleic acid amplification reaction (e.g., an RT-RamDA reaction) can be carried out with high amplification efficiency for a long period of time even in the presence of an amplification inhibitor such as a hydrophilic polymer. Thus, from yet another aspect, the present invention provides a kit for use in nucleic acid amplification, which includes a step of incubating at a temperature higher than 30° C. and lower than 50° C. for a period of time longer than 30 minutes.
In one embodiment, the kit includes a hydrophilic polymer and a single-stranded DNA binding protein at a concentration adjusted to give a final concentration of 40 ng/μL or more in a nucleic acid amplification reaction solution. Here, the concentration adjusted to give a final concentration of 40 ng/μL or more in a nucleic acid amplification reaction solution means, for example, that the reagent contains the single-stranded DNA binding protein at a concentration of 80 ng/μL or more when the reagent is diluted 2-fold for use, and that the reagent contains the single-stranded DNA binding protein at a concentration of 120 ng/μL or more when the reagent is diluted 3-fold for use.
In another embodiment, the kit comprises a hydrophilic polymer and 0.01 to 1500 parts by weight of a single-stranded DNA binding protein per 1 part by weight of the hydrophilic polymer.
The kit may be provided in a form in which both the hydrophilic polymer and the single-stranded DNA binding protein are filled in one container, or in a form in which each component is filled in a separate container and mixed at the time of use. The kit may also contain other components such as DNA strand-specific RNA:DNA hybrid strand decomposition enzyme, reverse transcriptase, primer, template nucleic acid, buffer, salt, etc. Specific types of components that can be included in the kit may be the same as those detailed in the above method. In addition, reaction conditions such as reaction temperature and reaction time of incubation performed when performing a nucleic acid amplification reaction using the kit may also be the same as those in the above method. By using the kit, it is possible to perform a long-term nucleic acid amplification reaction while suppressing a decrease in amplification efficiency, so that a large amount of cDNA can be amplified and a wide range of gene analysis can be performed.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Example 1: Effect of polyinosinic acid on long-term amplification in the RT-RamDA method In this example, the following experiment was performed to compare how the amplification rate in the RT-RamDA method changes with the addition of a nucleic acid polymer (polyinosinic acid) when the reaction time at 37°C is 2 hours.
To analyze the amplification rate in the RT-RamDA method, a nucleic acid fragment sample was first provided as a control in 5 μL of reverse transcription reaction solution 1 (normal reverse transcription reaction) shown in Table 1, and a reverse transcription reaction was carried out in reaction cycle 1 shown in Table 2. Separately, a nucleic acid fragment sample was provided in 5 μL of various RT-RamDA reaction solutions 1 (conditions 1 to 4) shown in Table 3, and an RT-RamDA reaction was carried out in reaction cycle 2 shown in Table 4. As the nucleic acid fragment sample, 10 pg of RNA purified from HeLa cells using RNeasy Mini Kit (Qiagen) was used.

Then, the samples were analyzed by real-time PCR according to the following method.
Specifically, the reaction solution after the reverse transcription reaction or the RT-RamDA reaction was diluted 10-fold with nuclease-free water (Qiagen), and 2 μL (1/25 volume) of the diluted solution was used for the qPCR reaction. qPCR was performed using StepOne Plus (Life Technologies) under the following conditions.
The qPCR reaction solution [20 μl (THUNDERBIRD™ SYBR qPCR Mix (TOYOBO), 6 pmol forward primer, 6 pmol reverse primer, 2 μl diluted reverse transcription reaction solution or RT-RamDA reaction solution, nuclease free water)] was treated at 95° C. for 1 minute to activate the enzyme, and then 40 cycles of denaturation at 95° C. for 15 seconds and extension reaction at 60° C. for 1 minute were performed.
Melting curve analysis was performed at 95°C for 15 seconds, 60°C for 15 seconds, and 95°C for 15 seconds.
As a target gene, the amplification rate of the βACTIN gene was analyzed by real-time PCR.
The primer set is as follows: (5'→3')
Forward primer: CGCGAGAAGATGACCCAGAT (SEQ ID NO: 1)
Reverse primer: GCCAGAGGCGTACAGGGGATA (SEQ ID NO: 2)
After the analysis, the Ct value of the RT-RamDA method obtained in the sample to which the RT-RamDA reaction solution 1 was added was subtracted from the Ct value of the normal reverse transcription reaction obtained in the sample to which the reverse transcription reaction solution 1 was added to obtain the ΔCt value, and the amplification rate by the RT-RamDA reaction was evaluated. Since the RT-RamDA method has a higher amplification rate than the normal reverse transcription reaction, the Ct value is lower than that of the normal reverse transcription reaction. Therefore, the higher the ΔCt value, the smaller the Ct value of each test reaction solution (each RT-RamDA reaction solution) is, which means that the amplification rate is higher.

The results of Example 1 are shown in Figure 1. It was found that when the reaction time was 120 minutes at 37°C, the amplification rate by RT-RamDA increased as the amount of polyinosinic acid added was reduced. This shows that the addition of a nucleic acid polymer (hydrophilic polymer) such as polyinosinic acid inhibits amplification when the RT-RamDA reaction is incubated for a long period of time.

Example 2: Evaluation of components that suppress long-term amplification inhibition by polyinosinic acid in the RT-RamDA method 1
In this example, the following experiment was performed to examine whether increasing the concentration of reverse transcriptase or single-stranded DNA binding protein would affect the inhibition of long-term amplification reaction of the RT-RamDA reaction by polyinosinic acid seen in Example 1.
To analyze the amplification rate in the RT-RamDA method, first, as a control, a nucleic acid fragment sample was provided in 5 μL of reverse transcription reaction solution 1 (normal reverse transcription reaction) shown in Table 1 as in Example 1, and a reverse transcription reaction was carried out in the reaction cycle shown in Table 2. Separately, a nucleic acid fragment sample was provided in 5 μL of various RT-RamDA reaction solutions 2 (conditions 1 to 4) shown in Table 5, and an RT-RamDA reaction was carried out in reaction cycle 3 shown in Table 6. As the nucleic acid fragment sample, 10 pg of RNA purified from HeLa cells using RNeasy Mini Kit (Qiagen) was used.

After the reverse transcription reaction or the RT-RamDA reaction, the amplification rate of the RT-RamDA reaction was analyzed by real-time PCR using the βACTIN gene and the NEAT1 gene as indicators in the same manner as in Example 1. The primer set used for the βACTIN gene was the same as that used in Example 1, and the following primer set was used for the NEAT1 gene.
Forward primer: CAGTTAGTTTATCAGTTCTCCCATCCA (SEQ ID NO: 3)
Reverse primer: GTTGTTGTCGTCACCTTTCAACTCT (SEQ ID NO: 4)
The reaction solution and thermal cycle conditions for real-time PCR were the same as those in Example 1.
After analysis, the amplification rate by the RT-RamDA reaction was evaluated by subtracting the Ct value of the RT-RamDA method obtained in the sample to which RT-RamDA reaction solution 2 was added from the Ct value of the normal reverse transcription reaction obtained in the sample to which reverse transcription reaction solution 1 was added, and calculating the ΔCt value.

The results of Example 2 are shown in Figure 2. Under conditions containing polyinosinic acid (condition 1), the amplification rate did not improve even when the reaction time at 37°C was extended beyond 30 minutes, and in some cases even decreased. Under conditions in which the reverse transcriptase was doubled in concentration (condition 2), the results were similar to condition 1, and no effect of suppressing the inhibition of amplification in long-term reactions was observed. On the other hand, under conditions in which the single-stranded DNA-binding protein T4 Gene 32 protein was doubled in concentration (conditions 3 and 4), the amplification rate tended to improve in proportion to the reaction time. Thus, it was shown that even when polyinosinic acid (hydrophilic polymer) was added, the amplification rate of the RT-RamDA reaction could be improved in proportion to the reaction time by increasing the amount of single-stranded DNA-binding protein.

Example 3: Evaluation of components that suppress long-term amplification inhibition by polyinosinic acid in the RT-RamDA method 2
In this example, it was examined whether the effect of increasing the concentration of the single-stranded DNA binding protein seen in Example 2 could be similarly confirmed when other reverse transcriptases were used.
As in Example 2, a nucleic acid fragment sample was first provided as a control in 5 μL of reverse transcription reaction solution 1 (normal reverse transcription reaction) shown in Table 1, and a reverse transcription reaction was carried out in the reaction cycle shown in Table 2. Separately, a nucleic acid fragment sample was provided in 5 μL of RT-RamDA reaction solution 3 (conditions 1 to 4) shown in Table 7, and an RT-RamDA reaction was carried out in reaction cycle 3 shown in Table 6, as in Example 2. As the nucleic acid fragment sample, 10 pg of RNA purified from HeLa cells using RNeasy Mini Kit (Qiagen) was used.

After the reverse transcription reaction or the RT-RamDA reaction, the amplification rate of the RT-RamDA reaction was analyzed by real-time PCR using the βACTIN gene and the NEAT1 gene as indicators in the same manner as in Example 1. The same primer sets for the βACTIN gene and the NEAT1 gene as in Example 2 were used.
The reaction solution and thermal cycle conditions for real-time PCR were the same as those in Example 1.
After analysis, the amplification rate by the RT-RamDA reaction was evaluated by subtracting the Ct value of the RT-RamDA method obtained in the sample to which RT-RamDA reaction solution 3 was added from the Ct value of the normal reverse transcription reaction obtained in the sample to which reverse transcription reaction solution 1 was added, and calculating the ΔCt value.

The results of Example 3 are shown in Figure 3. The same results were obtained when the reverse transcriptase was changed from ReverTra Ace (RT) manufactured by Toyobo Co., Ltd., used in Example 2, to PrimeScript (PS) manufactured by Takara Bio Inc. This further supports the idea that the amplification rate of the RT-RamDA reaction can be improved in proportion to the reaction time by increasing the amount of single-stranded DNA binding protein, even when polyinosinic acid (hydrophilic polymer) is added.

Example 4: Effect of concentration of single-stranded DNA binding protein In this example, the relationship between the amplification rate of the RT-RamDA reaction in the case of a long-term reaction and the concentration of the single-stranded DNA binding protein was analyzed in more detail.
As in the previous Examples, a nucleic acid fragment sample was first provided as a control in 5 μL of reverse transcription reaction solution 1 (normal reverse transcription reaction) shown in Table 1, and a reverse transcription reaction was carried out in the reaction cycle shown in Table 2. Separately, a nucleic acid fragment sample was provided in 5 μL of RT-RamDA reaction solution 4 (conditions 1 to 6) shown in Table 8, and an RT-RamDA reaction was carried out in reaction cycle 4 shown in Table 9. As the nucleic acid fragment sample, 10 pg of RNA purified from HeLa cells using RNeasy Mini Kit (Qiagen) was used.

After the reverse transcription reaction or the RT-RamDA reaction, the amplification rate of the RT-RamDA reaction was analyzed by real-time PCR using the βACTIN gene as an indicator in the same manner as in Example 1. The same primer set for the βACTIN gene as in Example 1 was used.
The reaction solution and thermal cycle conditions for real-time PCR were the same as those in Example 1.
After analysis, the Ct value of the RT-RamDA method obtained in the sample to which RT-RamDA reaction solution 4 was added was subtracted from the Ct value of the normal reverse transcription reaction obtained in the sample to which reverse transcription reaction solution 1 was added, and the amplification rate by the RT-RamDA reaction was evaluated by calculating the ΔCt value.

The results of Example 4 are shown in Figure 4. In a 120-minute RT-RamDA reaction, the amplification rate of the βACTIN gene improved in proportion to the T4 Gene 32 protein concentration. In particular, the amplification rate of the βACTIN gene improved significantly at T4 Gene 32 protein concentrations of 42 ng/μL or higher.

Example 5: Suppression effect against amplification inhibition by polycytidylic acid In this example, the effect of using polycytidylic acid (polyC), another hydrophilic polymer (nucleic acid polymer), in a long-term RT-RamDA reaction was examined.
As in the previous Examples, a nucleic acid fragment sample was first provided as a control in 5 μL of reverse transcription reaction solution 1 (normal reverse transcription reaction) shown in Table 1, and a reverse transcription reaction was carried out in the reaction cycle shown in Table 2. Separately, a nucleic acid fragment sample was provided in 5 μL of RT-RamDA reaction solution 5 (conditions 1 to 3) shown in Table 10, and an RT-RamDA reaction was carried out in reaction cycle 3 shown in Table 6. As the nucleic acid fragment sample, 10 pg of RNA purified from HeLa cells using RNeasy Mini Kit (Qiagen) was used.

After the reverse transcription reaction or the RT-RamDA reaction, the amplification rate of the RT-RamDA reaction was analyzed by real-time PCR using the βACTIN gene and the NEAT1 gene as indicators in the same manner as in Example 1. The same primer sets for the βACTIN gene and the NEAT1 gene as in Example 2 were used.
The reaction solution and thermal cycle conditions for real-time PCR were the same as those in Example 1.
After analysis, the Ct value of the RT-RamDA method obtained in the sample to which RT-RamDA reaction solution 5 was added was subtracted from the Ct value of the normal reverse transcription reaction obtained in the sample to which reverse transcription reaction solution 1 was added, and the amplification rate by the RT-RamDA reaction was evaluated by calculating the ΔCt value.

The results of Example 5 are shown in Figure 5. As in the previous Examples using polyinosinic acid, the amplification rate of the RT-RamDA reaction decreased in the samples to which polycytidylic acid was added in the RT-RamDA reaction over long reaction times of 120 and 360 minutes. Furthermore, by increasing the concentration of T4 Gene 32 protein, a single-stranded DNA binding protein, from 33 ng/μL to 67 ng/μL, the amplification rate of the RT-RamDA reaction over reaction times of 120 and 360 minutes improved. This shows that hydrophilic polymers other than polyinosinic acid also inhibit the RT-RamDA reaction, and that this inhibition can be alleviated by adding 40 ng/μL or more of a single-stranded DNA binding protein such as T4 Gene 32 protein.

Example 6: Suppression effect on amplification inhibition by polyethylene glycol In this example, the effect of using another hydrophilic polymer, polyethylene glycol (PEG), in a long-term RT-RamDA reaction was examined.
As in the previous Examples, a nucleic acid fragment sample was first provided as a control in 5 μL of reverse transcription reaction solution 1 (normal reverse transcription reaction) shown in Table 1, and a reverse transcription reaction was carried out in the reaction cycle shown in Table 2. Separately, a nucleic acid fragment sample was provided in 5 μL of RT-RamDA reaction solution 6 (conditions 1 to 3) shown in Table 11, and an RT-RamDA reaction was carried out in reaction cycle 3 shown in Table 6. As the nucleic acid fragment sample, 10 pg of RNA purified from HeLa cells using RNeasy Mini Kit (Qiagen) was used.

After the reverse transcription reaction or the RT-RamDA reaction, the amplification rate of the RT-RamDA reaction was analyzed by real-time PCR using the βACTIN gene as an indicator in the same manner as in Example 1. The same primer set for the βACTIN gene as in Example 1 was used.
The reaction solution and thermal cycle conditions for real-time PCR were the same as those in Example 1.
After analysis, the Ct value of the RT-RamDA method obtained in the sample to which RT-RamDA reaction solution 6 was added was subtracted from the Ct value of the normal reverse transcription reaction obtained in the sample to which reverse transcription reaction solution 1 was added, and the amplification rate by the RT-RamDA reaction was evaluated by calculating the ΔCt value.

The results of Example 6 are shown in Figure 6. A decrease in the amplification rate was also observed in the sample to which polyethylene glycol (PEG) was added, and the amplification rate of the RT-RamDA reaction was significantly decreased, especially in the RT-RamDA reactions over long periods of time, 120 and 360 minutes. In addition, by increasing the concentration of T4 Gene 32 protein, a single-stranded DNA binding protein, from 33 ng/μL to 67 ng/μL, the significant decrease in the amplification rate in the long-term RT-RamDA reactions over 120 and 360 minutes was significantly suppressed. Thus, it was shown that hydrophilic polymers other than nucleic acid polymers also inhibit the RT-RamDA reaction, and that this inhibition can be suppressed by adding 40 ng/μL or more of a single-stranded DNA binding protein such as T4 Gene 32 protein.

The present invention makes it possible to suppress the inhibitory effect even in systems where an inhibitor that reduces the amplification efficiency is added when a nucleic acid amplification reaction such as an RT-RamDA reaction is carried out for a long period of time at a specific temperature. This method is useful for various genetic analysis methods, such as single-cell analysis using the RT-RamDA method.

Claims (15)

A method for amplifying nucleic acid, comprising a step of incubating a nucleic acid amplification reaction solution containing a hydrophilic polymer at a temperature higher than 30°C and lower than 50°C for a time longer than 30 minutes, wherein the nucleic acid amplification reaction solution contains 40 ng/μL or more of a single-stranded DNA binding protein, thereby suppressing inhibition of nucleic acid amplification by the hydrophilic polymer. The method according to claim 1, in which nucleic acid is amplified by the RT-RamDA method. The method according to claim 1 or 2, wherein the amplification reaction solution contains a DNA strand-specific RNA:DNA hybrid strand degrading enzyme, 40 ng/μL or more of a single-stranded DNA binding protein, a reverse transcriptase, a primer, a template RNA, and a hydrophilic polymer. The method of claim 3, wherein the DNA strand-specific RNA:DNA hybrid strand degrading enzyme is a non-specific DNA degrading enzyme. The method according to claim 3 or 4, wherein the template RNA is RNA extracted from 1 cell to 1000 cells. The method according to any one of claims 1 to 5, wherein the single-stranded DNA binding protein is T4 gene 32 protein. The method according to any one of claims 1 to 6, wherein the incubation time is 60 minutes or more. The method according to any one of claims 1 to 7, wherein the incubation time is 120 minutes or more. The method according to any one of claims 1 to 8, wherein the hydrophilic polymer is at least one selected from the group consisting of a nucleic acid polymer and polyethylene glycol. The method according to any one of claims 1 to 9, wherein the concentration of the hydrophilic polymer in the nucleic acid amplification reaction solution is 0.01 ng/μL or more. The method according to any one of claims 1 to 10, wherein the hydrophilic polymer is at least one selected from the group consisting of polyinosinic acid and polycytidylic acid. The method according to any one of claims 1 to 11, wherein the incubation temperature is 35°C or higher and 45°C or lower. A method for amplifying nucleic acid, comprising a step of incubating a nucleic acid amplification reaction solution containing a hydrophilic polymer at a temperature higher than 30°C and lower than 50°C for a time longer than 30 minutes, wherein the nucleic acid amplification reaction solution contains 0.01 to 1500 parts by mass of a single-stranded DNA binding protein per part by mass of the hydrophilic polymer, thereby suppressing inhibition of nucleic acid amplification by the hydrophilic polymer. A kit for use in nucleic acid amplification, which includes a step of incubating a nucleic acid amplification reaction solution at a temperature higher than 30°C and lower than 50°C for a time longer than 30 minutes, the kit including a hydrophilic polymer and a single-stranded DNA binding protein at a concentration adjusted so that the concentration in the nucleic acid amplification reaction solution is 40 ng/μL or more. A kit for use in nucleic acid amplification, which includes a step of incubating a nucleic acid amplification reaction solution at a temperature higher than 30°C and lower than 50°C for a time longer than 30 minutes, the kit including a hydrophilic polymer and 0.01 to 1500 parts by mass of a single-stranded DNA binding protein per 1 part by mass of the hydrophilic polymer.