Patent Grant
10457982
The present invention relates to a method for amplifying a nucleic acid, in which a degrading enzyme specific to DNA in RNA-DNA hybrid and a strand displacement activity are used. More specifically, the present invention relates to a method for amplifying a nucleic acid, using RNA as a template, and also using a degrading enzyme specific to DNA in RNA-DNA hybrid and an RNase H minus reverse transcriptase. Moreover, the present invention relates to a kit used for performing the above-described method for amplifying a nucleic acid.
A large number of methods for amplifying a nucleic acid have been reported so far. Examples of such a method for amplifying a nucleic acid include Polymerase Chain Reaction (PCR) (Non Patent Literature 1), Strand Displacement Amplification (SDA) (Non Patent Literature 2), Multiple Displacement Amplification (MDA) (Non Patent Literature 3), Rolling-Circle Amplification (RCA) (Non Patent Literature 4), Loop-Mediated Isothermal Amplification (LAMP) (Non Patent Literature 5), Smart Amplification Process (SmartAmp) (Non Patent Literature 6), Helicase-Dependent Amplification (HDA) (Non Patent Literature 7), and Ligase Chain Reaction (LCR) (Non Patent Literature 8). These methods are amplification methods of using DNA as a template, and in all of the methods other than PCR, HDA and LCR, the strand displacement activity of DNA polymerase is utilized. A main amplification system of utilizing a strand displacement reaction is SDA (Non Patent Literature 2). In SDA, a cleavage site (nick) is made on one strand of double-stranded DNA using a restriction enzyme or the like. Using this nick as a starting point, the DNA strand on the 3′ side is peeled by the strand displacement activity of DNA polymerase, and novel complementary DNA is synthesized. SDA is a technique of amplifying complementary DNA by continuously generating this reaction. Moreover, MDA, which utilizes a random hexamer primer, is able to randomly take place a strand displacement reaction by randomly annealing primers to multiple sites on template DNA, and thus, this is a method having an extremely high amplification rate (Non Patent Literature 3). However, in order to amplify an RNA sequence according to these amplification methods using DNA as a template, it is necessary to perform a reverse transcription reaction of converting RNA to DNA. In addition, in the case of SDA, since a nick needs to be inserted into template DNA using restriction enzymes, it is necessary to add a restriction enzyme recognition sequence, deoxyinosine and the like to the template. Moreover, in the case of LAMP and SmartAmp, four or five sequence-specific primers are necessary for a single target. Thus, the synthesis of a special oligo primer, the designing of a sequence-specific primer, and the like become necessary. Furthermore, amplification methods using DNA as a template, such as PCR as a representative example, are problematic in terms of pseudo-positive results generated as a result of the carry-over of the previous PCR amplification products or reverse transcription products on samples, and non-specific products derived from DNA contaminated with reagents, DNA mixed from working environment, etc. (Non Patent Literatures 9 and 10). In particular, in the case of MDA, since primers are randomly annealed to DNA, it is likely that such contaminated DNA-derived non-specific products would be increased. As an amplification technique of using RNA as a template, a method of utilizing a special DNA hairpin primer having a restriction enzyme recognition sequence has been proposed. In this method, a cleavage point is generated on a DNA hairpin primer that has been allowed to bind to RNA as a template, and then, a strand displacement reaction is allowed to take place, so as to amplify complementary strand DNA. In this method, however, a pre-treatment needs to be performed to ligate a DNA hairpin primer to the 3′-end of template RNA, and thus, molecules that cannot be captured are likely to be generated depending on ligation conditions. Further, since amplification is started from the 3′-end of template RNA, it is considered difficult for this method to equally amplify the entire-length DNA.
Non Patent Literatures
To perform a gene expression analysis, it is necessary to convert RNA to complementary DNA (cDNA). There has been an amplification technique of using DNA as a template, but there have been no amplification techniques of directly using, as a template, untreated RNA that has not been subjected to a pre-treatment such as addition of a special sequence. It is an object of the present invention to provide, not the existing amplification method of using DNA as a template, but a method for amplifying a nucleic acid, using RNA as a template. That is, it is an object of the present invention to provide a method for amplifying a nucleic acid, using RNA as a template, which can realize not only simplification of operations, but also elimination of the risk of non-specific amplification caused by DNA contaminated with reagents and/or working environment, an increase in the detection sensitivity of trace RNA, and a reduction in amplification bias.
As a result of intensive studies directed towards achieving the aforementioned object, the present inventors have discovered a reverse transcription method with random displacement amplification (hereinafter also referred to as a “RT-RamDA method,” or simply a “RamDA method”), in which cDNA is amplified using RNA as a template. It have been revealed that, according to the RT-RamDA method, the yield of cDNA can be increased to 10 to 100 times as compared to a commercially available kit, and that it becomes possible to capture low expressed genes or to increase the number of detected genes in a gene expression analysis from a trace amount of RNA. Moreover, the cDNA amplified by the RamDA method can be applied to various gene analyses such as an RNA sequence method, and thus, it is expected that the RamDA method will be used as a basic technique of molecular biology in a wide range of fields. Furthermore, differing from the existing amplification technology of using DNA as a template, the use of RNA as a template can realize not only simplification of operations, but also elimination of the risk of non-specific amplification caused by DNA contaminated with reagents and/or working environment, and a reduction in amplification bias. The present invention has been completed based on these findings.
Specifically, according to the present invention, the following inventions are provided.
(1) A method for amplifying a nucleic acid, comprising a step of incubating a mixture containing template RNA, a primer, a degrading enzyme specific to DNA in RNA-DNA hybrid, an RNase H minus reverse transcriptase, and a substrate, wherein the degrading enzyme specific to DNA in RNA-DNA hybrid has an activity of cleaving a DNA strand in the RNA-DNA hybrid.
(2) The method for amplifying a nucleic acid according to (1), which comprises a step of synthesizing a complementary strand DNA (cDNA) of the template RNA by the RNA-dependent DNA polymerase activity of the RNase H minus reverse transcriptase, then randomly cleaving the cDNA strand in the hybrid strand of RNA and cDNA by the degrading enzyme specific to DNA in RNA-DNA hybrid, then peeling the cDNA strand on the 3′ side from the RNA by the strand displacement activity of the RNase H minus reverse transcriptase, while using the cleavage site as a starting point, and then synthesizing a novel cDNA strand in a portion peeled by the RNase H minus reverse transcriptase.
(3) The method for amplifying a nucleic acid according to (1) or (2), wherein the mixture comprises a double strand-specific DNA degrading enzyme as a degrading enzyme specific to DNA in RNA-DNA hybrid, and the double strand-specific DNA degrading enzyme has an activity of cleaving the DNA strand in the RNA-DNA hybrid, and substantially does not have an activity of cleaving the RNA strand in the RNA-DNA hybrid, single-stranded DNA and single-stranded RNA.
(4) The method for amplifying a nucleic acid according to (1) or (2), wherein the mixture comprises a non-specific DNA degrading enzyme as a degrading enzyme specific to DNA in RNA-DNA hybrid, and the non-specific DNA degrading enzyme has an activity of cleaving the DNA strand of the RNA-DNA hybrid strand, and substantially does not have an activity of cleaving the RNA strand in the RNA-DNA hybrid, and single-stranded RNA.
(5) The method for amplifying a nucleic acid according to any one of (1) to (4), wherein the degrading enzyme specific to DNA in RNA-DNA hybrid has a DNA-degrading activity even at a temperature of lower than 60° C.
(6) The method for amplifying a nucleic acid according to (3), wherein the double strand-specific DNA degrading enzyme is a double strand-specific DNA degrading enzyme derived from Crustacea, or a variant thereof.
(7) The method for amplifying a nucleic acid according to (6), wherein the double strand-specific DNA degrading enzyme is a double strand-specific DNA degrading enzyme derived from a shrimp, or a variant thereof.
(8) The method for amplifying a nucleic acid according to (4), wherein the non-specific DNA degrading enzyme is a non-specific DNA degrading enzyme derived from a mammal, or a variant thereof.
(9) The method for amplifying a nucleic acid according to (8), wherein the non-specific DNA degrading enzyme is a non-specific DNA degrading enzyme derived from a bovine, or a variant thereof.
(10) The method for amplifying a nucleic acid according to any one of (1) to (9), wherein the primer is one or more of a random primer, an oligo dT primer and a sequence-specific primer.
(11) The method for amplifying a nucleic acid according to any one of (1) to (10), wherein the primer is a primer, the Tm value of which is increased by modification with a cation unit.
(12) The method for amplifying a nucleic acid according to any one of (1) to (11), wherein the primer is a Zip Nucleic Acid (ZNA) primer.
(13) The method for amplifying a nucleic acid according to any one of (1) to (12), wherein the mixture further comprises a single-stranded DNA-binding protein.
(14) The method for amplifying a nucleic acid according to any one of (1) to (13), wherein the template RNA is trace RNA corresponding to a volume ranging from a single cell to several hundreds of cells.
(15) The method for amplifying a nucleic acid according to any one of (1) to (14), which is performed to amplify cDNA that is to be subjected to the production of a DNA sequence library.
(16) A kit used for performing the method for amplifying a nucleic acid according to any one of (1) to (15), wherein the kit comprises, at least, a degrading enzyme specific to DNA in RNA-DNA hybrid and an RNase H minus reverse transcriptase.
(17) The kit according to (16), which further comprises a single-stranded DNA-binding protein.
Since a nucleic acid can be amplified using RNA as a template according to the method for amplifying a nucleic acid of the present invention, the present method can realize not only simplification of operations, but also elimination of the risk of non-specific amplification caused by DNA contaminated with reagents and/or working environment, and a reduction in amplification bias. Moreover, according to the method for amplifying a nucleic acid of the present invention, the yield that is 10 times or more the yield as compared to the existing reverse transcription cDNA synthesis kit can be obtained, and it is also possible to perform amplification at a magnification of 100 times or more, depending on conditions.
Hereinafter, the present invention will be more specifically described.
The method for amplifying a nucleic acid according to the present invention is carried out by incubating a mixture comprising template RNA, a primer, a degrading enzyme specific to DNA in RNA-DNA hybrid an RNase H minus reverse transcriptase, and a substrate, and this method is also referred to as Reverse Transcription with Random Displacement Amplification (RT-RamDA) Method. The method for amplifying a nucleic acid of the present invention is a reverse transcription method with random displacement amplification, in which a strand displacement reaction is utilized and RNA is directly used as a template, and this method can be carried out in vitro. The method of the present invention is a technique of amplifying cDNA in a reverse transcription reaction, whereby the cDNA strand in the RNA-cDNA hybrid is cleaved by utilizing the nuclease activity of a degrading enzyme specific to DNA in RNA-DNA hybrid as endonuclease, such as a double strand-specific DNA degrading enzyme (double-strand specific DNase: dsDNase) or a non-specific DNA degrading enzyme (DNase I), so that a strand displacement reaction is allowed to randomly take place.
In the present description, the term “amplification” is used to mean to increase the number of copies (replications) of the sequence of a nucleic acid. In addition, examples of the form of amplification include linear amplification, real-time amplification, quantitative amplification, semi-quantitative amplification, and competitive amplification, but are not particularly limited thereto.
The mixture used in the present invention is a mixture comprising template RNA, a primer, a degrading enzyme specific to DNA in RNA-DNA hybrid, an RNase H minus reverse transcriptase, and a substrate. The present mixture is preferably an aqueous buffer, and more preferably an aqueous buffer comprising salts.
An outline of one aspect of the RT-RamDA method of the present invention will be described below (
1. The complementary DNA (cDNA) of template RNA is synthesized by the RNA-dependent DNA polymerase activity of an RNase H minus reverse transcriptase.
2. A cleavage point (nick) is randomly made in the cDNA strand in the RNA-cDNA hybrid by the nuclease activity of a degrading enzyme specific to DNA in RNA-DNA hybrid, such as a double strand-specific DNA degrading enzyme (dsDNase) or a non-specific DNA degrading enzyme (DNase I).
3. The nick site becomes a starting point, and the cDNA strand on the 3′ side is peeled by the strand displacement activity of the RNase H minus reverse transcriptase. cDNA is newly synthesized in the peeled portion by reverse transcriptase. The peeled cDNA is protected from the nuclease activity by a T4 gene 32 protein (T4GP32) as a single-stranded DNA-binding protein. When this phenomenon takes place continuously, the yield of cDNA can be increased to 10-fold or more.
Since the RNA-DNA hybrid strand is targeted by the degrading enzyme specific to DNA in RNA-DNA hybrid in the method for amplifying a nucleic acid of the present invention, or since the template is RNA in the strand displacement amplification reaction, the available reverse transcriptase is limited to those having no RNase H activity. The RNase H activity is ribonuclease activity of randomly cleaving only the RNA strand in an RNA-DNA hybrid. RNase H is non-specific endonuclease, which catalyzes RNA cleavage by hydrolysis.
Moreover, the RNase H minus reverse transcriptase used in the present invention has a strand displacement activity. The cDNA strand on the 3′ side is peeled from RNA by the strand displacement activity of the RNase H minus reverse transcriptase, and a new cDNA strand is synthesized in the peeled portion.
The reverse transcription is a process in which reverse transcriptase (RNA-dependent DNA polymerase) catalyzes the formation of complementary DNA (cDNA) from template RNA. As such reverse transcriptase, a large number of enzymes have been identified, and examples of the reverse transcriptase include HIV reverse transcriptase, AMV reverse transcriptase, M-MLV reverse transcriptase, C therm. polymerase, and Tth polymerase. In the present invention, RNase H minus reverse transcriptase is used.
In the present invention, a degrading enzyme specific to DNA in RNA-DNA hybrid is used. The term “degrading enzyme specific to DNA in RNA-DNA hybrid” is used in the present description to mean an enzyme having an activity of cleaving a DNA strand in a hybrid strand of RNA and DNA (RNA-DNA hybrid). As such a degrading enzyme specific to DNA in RNA-DNA hybrid, a double strand-specific DNA degrading enzyme or a non-specific DNA degrading enzyme can be used. As a double strand-specific DNA degrading enzyme, a crab-derived double strand-specific DNA degrading enzyme (double strand-specific nuclease; DSN) has been known. This enzyme randomly cleaves not only double stranded DNA, but also only the DNA strand in an RNA-DNA hybrid. In the present invention, there has been proposed a method of utilizing the aforementioned activity to cleave the cDNA in an RNA-cDNA hybrid formed during reverse transcription, so as to cause strand displacement amplification while using RNA as a template. However, since the temperature for activation of crab-derived DSN is an extremely high temperature that is 60° C. or higher, it is unsuitable for a general reverse transcription reaction (Non Patent Literature 11). Hence, by using shrimp-derived heat-labile dsDNase that is active even at a low temperature, both a reverse transcription reaction and cleavage with DNase could be achieved (Non Patent Literature 12). On the other hand, when a non-specific DNA degrading enzyme such as DNase I is used, single-stranded DNA, namely, amplified DNA that is peeded by a strand displacement reaction or a reverse transcription primer is likely to be decomposed. However, even if the enzyme has an activity of degrading single-stranded DNA, it is a case where it would not cause a problem, depending on reaction conditions such as the composition of a buffer used. Whether or not the enzymes used or reaction conditions are suitable can be confirmed by performing a nucleic acid amplification reaction according to the disclosure of the present description. Moreover, by addition of a single-stranded DNA-binding protein, the degradation activity of single-stranded DNA is suppressed, and the synthesis and amplification of cDNA can be promoted by a strand displacement reaction using reverse transcriptase. As such a single-stranded DNA-binding protein, any given protein, which binds to single-stranded DNA to increase the efficiency of nucleic acid amplification according to the method of the present invention, can be used, and for example, any given origin-derived T4 gene 32 protein, RecA, SSB (Single-Stranded DNA Binding Protein), or a variant thereof can be used. Thereby, the amplification rate can be increased, rather than by a method of using dsDNase. The use of dsDNase or DNase I is advantageous in that DNA contaminated with the reaction solution, for example, genomic DNA in a cell lysate sample can also be removed. Also in the past, there have been reported methods of removing contaminated DNA using DNase I or dsDNase in RT-qPCR. However, in a majority of such methods, inactivation of nuclease has been essential, and thus, such methods could not be carried out by only one step consisting of a reverse transcription reaction, like the RT-RamDA method (Non Patent Literature 13).
There have been many reports regarding DNA amplification methods using a strand displacement reaction, as with the RT-RamDA method (for example, Non Patent Literatures 2 to 6). However, a majority of these amplification methods including SDA as a typical example need to use restriction enzymes to insert a nick into template DNA, or need to use a plurality of sequence-specific primers in combination. Accordingly, it is essential to add a restriction enzyme recognition sequence or deoxyinosine to template DNA, or to design and/or synthesize a sequence-specific primer. Moreover, target genes are also limited. Furthermore, since almost all of existing methods use DNA as a template, it is necessary to perform a reverse transcription reaction of converting RNA to DNA in order to amplify an RNA sequence. Further, pseudo-positive results generated as a result of the carry-over of the previous PCR amplification products or reverse transcription products on samples, and non-specific products derived from DNA contaminated with reagents, DNA mixed from working environment, etc., have been problematic (Non Patent Literatures 9 and 10). In particular, in MDA, since a primer is randomly annealed to DNA, it is highly likely that the contaminated DNA-derived non-specific products would be increased. On the other hand, the RT-RamDA method of the present invention uses a non-sequence-dependent strand displacement reaction, and thus, it becomes unnecessary to design and/or synthesize a sequence-specific primer or a special primer, and also, it can target whole genes. Furthermore, by directly using RNA as a template, the process from reverse transcription to amplification can be completed by only one step, and since DNA hardly becomes a template, amplification of non-specific products derived from the contaminated DNA can also be reduced.
The double strand-specific DNA degrading enzyme used in the present invention can be preferably an enzyme, which has an activity of cleaving the DNA strand of an RNA-DNA hybrid, and substantially does not have an activity of cleaving the RNA strand of an RNA-DNA hybrid, single-stranded DNA and single-stranded RNA. The double strand-specific DNA degrading enzyme is preferably an enzyme having a DNA-degrading activity even at a temperature of lower than 60° C.
As such double strand-specific DNA degrading enzyme, enzymes derived from prokaryotes or eukaryotes can be used. Preferably, a Crustacea-derived double strand-specific DNA degrading enzyme or a variant thereof can be used.
As such double strand-specific DNA degrading enzyme (which is also referred to as “double strand-specific nuclease (DSN)”), the following enzymes have been known so far (JP Patent Publication (Kokai) 2014-103867 A).
(1) Solenocera melantho (coastal mud shrimp) DNase
(2) Penaeus japonicus (prawn) DNase
(3) Paralithodes camtschaticus (king crab) DSN
(4) Pandalus borealis (Northern shrimp) dsDNase
(5) Chionoecetes opilio (snow crab) DSN
(6) Other DSN homologs
Among the above-described enzymes, king crab DSN and snow crab DSN have heat resistance at an optimum active temperature of about 60° C., whereas Northern shrimp dsDNase is a heat-labile enzyme having an optimum temperature of 37° C.
In the present invention, a shrimp-derived double strand-specific DNA degrading enzyme or a variant thereof is further preferable.
In the present description, the term “variant” is used to mean an enzyme obtained by modifying the amino acid sequence of a natural product-derived double strand-specific DNA degrading enzyme. Specifically, such a variant means an enzyme consisting of an amino acid sequence having sequence identity of 80% or more (preferably 90% or more, and more preferably 95% or more) to the amino acid sequence of a natural product-derived double strand-specific DNA degrading enzyme, and having a double strand-specific DNA-degrading activity, and also, an enzyme consisting of an amino acid sequence comprising a deletion, substitution and/or addition of one or several amino acids (for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids) with respect to the amino acid sequence of a natural product-derived double strand-specific DNA degrading enzyme, and having a double strand-specific DNA-degrading activity.
As a double strand-specific DNA degrading enzyme, a commercially available product can be used. Examples of such a commercially available product include dsDNase (ArcticZymes), Hl-dsDNase (ArcticZymes), dsDNase (Thermo scientific), Shrimp DNase, Recombinant (Affymetrix), Atlantis dsDNase (Zymo Research), and Thermolabile Nuclease (Roche).
An example of the non-specific DNA degrading enzyme used in the present invention is an enzyme, which has an activity of cleaving the DNA strand of an RNA-DNA hybrid and substantially does not have an activity of cleaving the RNA strand of an RNA-DNA hybrid and single-stranded RNA, wherein the activity of cleaving the single-stranded DNA is preferably lower than the activity of cleaving the DNA strand of the RNA-DNA hybrid. The non-specific DNA degrading enzyme is preferably an enzyme having a DNA-degrading activity even at a temperature of lower than 60° C.
As such a non-specific DNA degrading enzyme, an enzyme derived from prokaryotes or eukaryotes can be used. Preferably, a mammal-derived non-specific DNA degrading enzyme or a variant thereof can be used.
In the present invention, a bovine-derived non-specific DNA degrading enzyme or a variant thereof is more preferable.
In the present description, the term “variant” is used to mean an enzyme obtained by modifying the amino acid sequence of a natural product-derived non-specific DNA degrading enzyme. Specifically, the variant means an enzyme consisting of an amino acid sequence having sequence identity of 80% or more (preferably 90% or more, and more preferably 95% or more) to the amino acid sequence of a natural product-derived non-specific DNA degrading enzyme, and having a non-specific DNA-degrading activity, and also, an enzyme consisting of an amino acid sequence comprising a deletion, substitution and/or addition of one or several amino acids (for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids) with respect to the amino acid sequence of a natural product-derived non-specific DNA degrading enzyme, and having a non-specific DNA-degrading activity.
The primer used in the present invention is composed of a deoxynucleotide and/or a ribonucleotide, and the present primer has a chain length capable of undergoing base pairing with a target nucleic acid under given conditions. The chain length of such a primer is not particularly limited, and it is preferably 5 to 50 nucleotides, and more preferably 5 to 30 nucleotides. As such primers, one or more of types of random primers, oligo dT primers or sequence-specific primers can be preferably used. In the case of using a random primer, the length of the primer is preferably approximately 5 to 10 nucleotides, and more preferably approximately 6 to 8 nucleotides. In the case of using an oligo dT primer, the length of the primer is preferably 10 to 50 nucleotides, and more preferably 15 to 30 nucleotides. In the case of using a sequence-specific primer, the length of the primer is preferably 5 to 30 nucleotides, and more preferably 7 to 20 nucleotides.
The primer can be synthesized by any given method that can be used in the synthesis of an oligonucleotide, such as a phosphite triester method, an H-phosphonate method, or a thiophosphonate method. The primer according to the present invention can be synthesized, for example, by a phosphoramidite method, using DNA Synthesizer Type 394 manufactured by ABI (Applied Biosystem Inc.).
Preferably, a primer, the Tm value of which has been increased by modification with a cation unit, can be used. For example, a Zip Nucleic Acid (ZNA) primer can be used. ZNA has an action to increase the Tm value of an oligonucleotide, using a cation unit (Non Patent Literatures 14 to 16). By using a primer, the Tm value of which has been increased by modification with a cation unit, such as a ZNA primer, amplification efficiency can be enhanced. In particular, by using a random hexamer primer modified with ZNA, the primer can be annealed to template RNA even at a reaction temperature during reverse transcription, and thereby, more efficient strand displacement amplification can be carried out.
The term “substrate” is used in the present description to mean a substrate of RNase H minus reverse transcriptase, and as such a substrate, a mixture of four types of deoxyribonucleotides (dATP, dCTP, dGTP, and dTTP) can be used. However, the substrate is not limited to the mixture of dATP, dCTP, dGTP, and dTTP, and dideoxynucleotide triphosphate may be added to the mixture, or a modified deoxyribonucleotide may also be used.
In the present invention, the mixture to be used in the reaction may comprise a single-stranded DNA-binding protein such as a T4 gene 32 protein. The T4 gene 32 protein can be used as an auxiliary factor for increasing the uniformity of amplification. The T4 gene 32 protein as a single-stranded DNA-binding protein has been known to act, not only on single-stranded DNA, but also on RNA (Non Patent Literatures 17 to 20). By using the T4 gene 32 protein, the higher structure of template RNA can be loosened, and a more uniform strand displacement reaction can be carried out on the full-length template. Moreover, in the case of a reaction using a non-specific DNA degrading enzyme, decomposition of amplified cDNA can be prevented.
The nucleic acid amplification reaction according to the present invention may be carried out under isothermal conditions, or under thermal cycling conditions.
When the nucleic acid amplification reaction is carried out under isothermal conditions, the reaction can be carried out, for example, at a predetermined temperature between 25° C. and 50° C., preferably at a predetermined temperature between 30° C. and 45° C., and more preferably at a predetermined temperature between 35° C. and 40° C., and thus, the reaction can be carried out, for example, at 37° C. for a predetermined period of time (for example, within 5 minutes to 3 hours, and preferably 10 minutes to 150 minutes). When the reaction is carried out, for example, at 37° C., after the reaction solution has been incubated, for example, at 25° C. for a predetermined period of time (for example, 5 minutes to 15 minutes), and then, has been incubated at 30° C. for a predetermined period of time (for example, 5 minutes to 15 minutes), the reaction is carried out at 37° C. Alternatively, after the reaction solution has been incubated at 37° C., it may be incubated at 50° C. for a predetermined period of time (for example, 5 minutes to 15 minutes), and then at 85° C. for a predetermined period of time (for example, 5 minutes to 15 minutes).
When the nucleic acid amplification reaction is carried out under thermal cycling conditions, for example, a predetermined temperature T1 of 20° C. or higher and lower than 30° C. (for example, 25° C.) is combined with a predetermined temperature T2 of 30° C. or higher and 45° C. or lower (for example, 37° C.). Then, T1 for a predetermined period of time (for example, 1 minute to 3 minutes, and as an example, 2 minutes) and T2 for a predetermined period of time (for example, 1 minute to 3 minutes, and as an example, 2 minutes) are set at one cycle, and this cycle is repeated preferably for 10 cycles to 40 cycles, and more preferably for 15 cycles to 35 cycles, so that the reaction can be carried out. In addition, prior to the above-described thermal cycling, the reaction solution may be incubated, for example, at 25° C. for a predetermined period of time (for example, 5 minutes to 15 minutes), then at 30° C. for a predetermined period of time (for example, 5 minutes to 15 minutes), and then at 37° C. for a predetermined period of time (for example, 1 minute to 5 minutes). Moreover, after completion of the above-described thermal cycling, the reaction solution may be incubated at 50° C. for a predetermined period of time (for example, 5 minutes to 15 minutes), and then at 85° C. for a predetermined period of time (for example, 5 minutes to 15 minutes).
The method for amplifying a nucleic acid according to the present invention can be used as a part of an RT-qPCR method of using a trace amount of RNA (for example, trace RNA corresponding to a volume ranging from a single cell to several hundreds of cells).
The method for amplifying a nucleic acid according to the present invention can be utilized in an RNA sequence method of using a trace amount of RNA (for example, trace RNA corresponding to a volume ranging from a single cell to several hundreds of cells).
By utilizing the method for amplifying a nucleic acid according to the present invention, it is possible to detect a single cell in a large amount of cells. Specifically, by specifically amplifying genes expressing only in target cells contained in a large amount of cells, it becomes possible to detect only a few target cell genes contained in a large amount of RNA, so that the presence or absence of target cells can be judged.
The present invention further relates to a kit for carrying out the above-described method for amplifying a nucleic acid according to the present invention. The kit of the present invention comprises, at least, a DNA strand-specific RNA-DNA hybrid strand degrading enzyme such as a double strand-specific DNA degrading enzyme or a non-specific DNA degrading enzyme, and an RNase H minus reverse transcriptase. Preferably, the kit of the present invention further comprises a single-stranded DNA-binding protein (for example, a T4 gene 32 protein). The kit of the present invention may further comprise, as desired, other reagents necessary for carrying out the nucleic acid amplification reaction, a buffer, and the like. Examples of such other reagents include a primer, and deoxyribonucleotide triphosphate.
The present invention will be described in more detail in the following examples. However, these examples are not intended to limit the scope of the present invention.
The RT-RamDA method is a method for amplifying a nucleic acid, which comprises a step of incubating a mixture comprising template RNA, a primer, a degrading enzyme specific to DNA in RNA-DNA hybrid such as a double strand-specific DNA degrading enzyme or a non-specific DNA degrading enzyme, an RNase H minus reverse transcriptase, and a substrate. In the RT-RamDA method, the complementary strand DNA (cDNA) of template RNA is synthesized by the RNA-dependent DNA polymerase activity of the RNase H minus reverse transcriptase, the cDNA strand in the RNA-cDNA hybrid is randomly cleaved with the degrading enzyme specific to DNA in RNA-DNA hybrid. The above described cleavage site becomes a starting point, the cDNA strand on the 3′ side is peeled from RNA by the strand displacement activity of the RNase H minus reverse transcriptase, and a novel cDNA strand is synthesized in the portion peeled by the RNase H minus reverse transcriptase.
RamDA-A is an aspect of carrying out the RT-RamDA method, using a double strand-specific DNA degrading enzyme and a T4 gene 32 protein.
RamDA-B is an aspect of carrying out the RT-RamDA method, using a double strand-specific DNA degrading enzyme, without using a T4 gene 32 protein.
RamDA-C is an RT-RamDA thermal cycling method, which is an aspect of performing a reaction by modifying reaction temperature conditions, while maintaining the same composition of the reaction solution as that in RamDA-B, and by repeating with short intervals an annealing step and an elongation reaction step.
RamDA-D is an aspect of carrying out the RT-RamDA method, using a non-specific DNA degrading enzyme and a T4 gene 32 protein.
(Materials and Methods)
Cell Culture
For extraction of total RNA, 5G6GR mouse ES cells were used. This cell line was produced by randomly incorporating a linearized Gata6-GR-IRES-Puro vector into EB5 ES cells (Non Patent Literature 20). The cells were cultured on a feeder-free gelatin-coated dish, in a 10% fetal bovine serum-containing Glasgow minimal essential medium (GMEM; Sigma-Aldrich, St Louis, Mo., USA) comprising 1000 U/ml leukemia inhibitory factor (ESGRO; Invitrogen Corp., Carlsbad, Calif., USA), 100 μmol/l 2-mercaptoethanol (Nacalai Tesque Inc., Kyoto, Japan), 1× non-essential amino acids (Life Technologies Corp., Carlsbad, Calif., USA), 1 mmol/1 sodium pyruvate (Life Technologies), 2 mmol/1 L-glutamine (Nacalai Tesque), 0.5× penicillin/streptomycin (Life Technologies), and 10 μg/ml blasticidin (Life Technologies).
RNA Extraction
Total RNA was purified using RNeasy Mini Kit (Qiagen Inc., Valencia, Calif., USA). Quantification of RNA and the quality check thereof were carried out using Quantus Fluorometer (Promega Corp., Madison, Wis., USA) and RNA 6000 Nano Kit (Agilent Biotechnology, Santa Clara, Calif., USA). Artificially synthesized RNA was synthesized from pGIBS-DAP, pGIBS-THR plasmid (American Type Culture Collection (ATCC), Manassas, Va., USA), using MEGAscript T3 kit (Life Technologies).
Preparation of 1-Cell Lysate of Mouse ES Cells
The cultured cells were reacted at 37° C. for 3 minutes, using TrypLE Express (Life Technologies), so as to dissociate the cells into 1 cell. After completion of the dissociation, the reagent was immediately replaced with PBS (−) to terminate the reaction. In order to precisely fractionate only the cell using a cell sorter, the living cell nucleus of the dissociated cell was labeled by staining it using Vybrant Dye Cycle (Life Technologies). The method included in the reagent was applied as staining conditions. Using SH 800 Cell Sorter (Sony Corp., Tokyo, Japan), cells, which showed positive to Vybrant Dye Cycle (detection filter: BP450/50) and showed negative to dead cell fluorescence marker dye, PI (detection filter: BP585/40), were defined as a living cell fraction. From this fraction, 50 cells were fractionated into 1 μl of Lysis buffer (1 U of RNasein plus (Promega), 0.3% NP40 (Thermo Fisher Scientific Inc., Waltham, Mass. USA), and RNase free water (TaKaRa Bio Inc., Otsu, Japan)). After completion of the fractionation, the cells were immediately subjected to spinning down and stirring by shaking, and the resulting cells were then conserved at −80° C. When the cells were used in a reverse transcription reaction, the cells were thawn, and 49 μl of Lysis buffer was then added to the resulting cells, so that 1 μl of 50-fold diluted cell lysate was used as a 1-cell lysate.
FRET Assay
FRET analysis was carried out by modifying the method of Inge W. Nilsen et al (Non Patent Literature 12). 0.6 μl of oligonucleotide probe mix (6 pmol of oligo DNA or RNA (Sigma-Aldrich), 1× First-Strand buffer (Life Technologies), and RNase free water (TaKaRa)) was prepared, and was then denatured at 70° C. for 5 minutes. Thereafter, the temperature was slowly returned to room temperature. Combinations of oligonucleotides used in the analysis are as follows (SEQ ID NOS: 1 to 10).
Subsequently, 5.4 μl of enzyme solution (1× First-Strand buffer, 0.12 U of each nuclease, and RNase free water) was prepared. As nucleases, DNase I Amplification Grade (Life Technologies), dsDNase (ArcticZymes AS, Tromso, Norway), and HL-dsDNase (ArcticZymes) were used. As a control, RNase free water was added instead of the enzyme. 0.6 μl of the oligonucleotide probe mix, which had been returned to room temperature, was mixed with 5.4 μl of the enzyme solution on PloxiPlate-384F Plus (PerkinElmer Inc., Waltham, Mass., USA), and the obtained mixture was then measured using EnVision (PerkinElmer). For the measurement, the fluorescence intensities of FAM and HEX were measured at 37° C. at a rate of one measurement for 70 seconds, and this measurement was carried out 50 times. The leakage of the fluorescence of FAM and HEX was corrected using the fluorescence amount of each single probe for correction.
In a verification experiment regarding the action of a T4 gene 32 protein on nuclease activity (
In a measurement experiment regarding KCl and Tris-HCl concentration-dependent nuclease activity of DNase I (
Reverse Transcription Reaction
RT-RamDA Method (RamDA-A):
Template RNA was diluted with 1 μl of Lysis buffer (1 U of RNasein plus (Promega), 10% Roche lysis buffer (Roche), 0.3% NP40 (Thermo Fisher), and RNase free water), and was then subjected to a denaturation treatment at 65° C. for 2 minutes. The resulting RNA was conserved on ice before use. ReverTra Ace qPCR RT KIT (TOYOBO Co. Ltd., Osaka, Japan) was modified, and was used as a reverse transcriptase the reaction solution. To 1 μl of the denatured diluted template RNA, 2 μl of RT mix (1.5× ReverTra Ace RT buffer (TOYOBO), 0.6 pmol of oligo (dT) 18 primer (Thermo Fisher), 7.8 pmol of random hexamer primer (TaKaRa), 1.5× ReverTra Ace enzyme mix (TOYOBO), and RNase free water), or 2 μl of RamDA-A mix (1.5× ReverTra Ace RT buffer, 0.6 pmol of oligo (dT) 18 primer, 7.8 pmol of ZNA-random hexamer primer ([Z][Z]NNNNNN; Sigma-Aldrich), 0.4 U of dsDNase (ArcticZymes), 100 ng of T4 gene 32 protein, 1.5× ReverTra Ace enzyme mix, and RNase free water) was added. The thus obtained mixtures were each stirred at 2,000 rpm for 30 seconds using MixMate (Eppendorf, Westbury, N.Y., USA), and were then reacted using a thermal cycler C1000 (Bio-Rad Laboratories, Inc., Hercules, Calif., USA)), at 25° C. for 10 minutes, at 30° C. for 10 minutes, at 37° C. for 30 minutes, at 50° C. for 5 minutes, and at 85° C. for 5 minutes.
RT-RamDA Method without Using T4 Gene 32 Protein (RamDA-B):
Template RNA was diluted with 1 μl of lysis buffer (1 U of RNasein plus, 0.3% NP40, and RNase free water), and was then subjected to a denaturation treatment at 70° C. for 90 seconds. The resulting RNA was cooled on ice, and thereafter, 2 μl of RT mix (1.5× ReverTra Ace RT buffer, 0.6 pmol of oligo (dT) 18 primer, 7.8 pmol of random hexamer primer, 1.5× ReverTra Ace enzyme mix, and RNase free water), or 2 μl of RamDA-B mix (1.5× ReverTra Ace RT buffer, 0.6 pmol of oligo (dT) 18 primer, 7.8 pmol of ZNA-random hexamer primer([Z][Z]NNNNNN; Sigma-Aldrich), and 0.1 U of dsDNase) was added. Thereafter, the thus obtained mixtures were each subjected to the same operations as those performed in RamDA-A.
RT-RamDA Thermal Cycling Method (RamDA-C):
A reverse transcription reaction was carried out with the same reaction solution composition as that of RamDA-B, with the exception that the temperature conditions were changed. The reaction conditions are as follows. After a reaction had been carried out at 25° C. for 10 minutes, at 30° C. for 10 minutes, and at 37° C. for 2 minutes, a reaction at 25° C. for 2 minutes, and then at 37° C. for 2 minutes was carried out for 29 cycles. Thereafter, the reaction product was treated at 50° C. for 5 minutes, and then at 85° C. for 5 minutes.
RT-RamDA Non-Specific DNA Degrading Enzyme (DNase I) Method (RamDA-D):
Template RNA was diluted with 1 μl of Lysis buffer (1 U of RNasein plus, 10% Roche lysis buffer, 0.3% NP40, and RNase free water), and was then subjected to a denaturation treatment at 70° C. for 90 seconds. The resulting RNA was conserved on ice before use. PrimeScript RT reagent Kit (Perfect Real Time) (TaKaRa) was modified, and was used as a reverse transcriptase the reaction solution. To 1 μl of the denatured template RNA, 2 μl of RT mix (1.5× PrimeScript Buffer (for Real Time), 0.6 pmol of oligo (dT) 18 primer, 8 pmol of random hexamer primer, 1.5× PrimeScript RT Enzyme Mix I, and RNase free water), or 2 μl of RamDA-D mix (1.5× PrimeScript Buffer (for Real Time), 0.6 pmol of oligo (dT) 18 primer, 8 pmol of random hexamer primer, 0.2 U of DNase I, Amplification Grade, 100 ng of T4 gene 32 protein, 1.5× PrimeScript RT Enzyme Mix I, and RNase free water) was added. The thus obtained mixtures were each reacted at 25° C. for 10 minutes, at 30° C. for 10 minutes, at 37° C. for 60 minutes, at 50° C. for 5 minutes, and at 85° C. for 5 minutes.
Experiment for Studying Composition of Reverse Transcription Reaction Solution, Using RamDA-D:
When a in-house reverse transcription reaction buffer, in which the concentrations of Tris-HCl, KCl, NaCl and MgCl2 were changed, or First-Strand Buffer was used, these were replaced with PrimeScript Buffer (for Real Time) in RamDA-D mix, and dNTP Mix (Life Technologies) was further added to the solution to a final concentration of 0.5 mM, followed by performing the reaction. The composition of each buffer is shown in Table 1 below.
Artificially Synthesized RNA (Dap and Thr) System of Using RamDA-D:
Only 0.6 pmol of oligo (dT) 18 primer was used as a reverse transcription primer in RamDA-D mix, and the composition of the RamDA-D mix was changed depending on conditions. In addition, the temperature conditions for the reverse transcription reaction were determined to be the same conditions as those for RamDA-A.
Studies Regarding Reverse Transcriptase in RamDA-D:
With regard to SuperScript II, SuperScript III, and SuperScript IV conditions, 30 U of SuperScript II (Life Technologies), 30 U of SuperScript III (Life Technologies), and 30 U of SuperScript IV (Life Technologies) were used as reverse transcriptase in RamDA-D mix, instead of PrimeScript RT Enzyme Mix I, and further, 6 U of RNasein plus were also added. As a reverse transcription reaction buffer, PrimeScript Buffer (for Real Time) was used for all conditions.
Comparison of dsDNase:
Nuclease was replaced with 0.2 U of 43 KDa dsDNase (ArcticZymes), 0.2 U of 47 KDa dsDNase (Affymetrix Inc., Santa Clara, Calif., USA), and 0.2 U of duplex specific nuclease (DSN; Evrogen JSC, Moscow, Russia), respectively, under the RamDA-A reaction solution conditions, and a comparison was then made. The reaction temperature conditions were determined in accordance with RamDA-A, and the amplification rate of cDNA was then quantified by qPCR.
Comparison of Reverse Transcriptase:
A comparison was made using 10 types of qPCR RT kits, Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher), ReverTra Ace qPCR RT Kit (TOYOBO), PrimeScript RT reagent Kit (TaKaRa), AffinityScript QPCR cDNA Synthesis Kit (Agilent Biotechnology), QuantiTect Rev. Transcription Kit (Qiagen), GoScript Reverse Transcription System (Promega), iScript Select cDNA Synthesis Kit (Bio-Rad), ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs, MA, UK), SuperScript III (Life Technologies), and Transcriptor First Strand cDNA Synthesis Kit (Roche).
For an elongation activity test, a reverse transcription reaction was carried out using Millennium RNA Markers (Life Technologies) comprising poly-A-added RNAs with various lengths (0.5-9 k bases). A reverse transcription mixture was prepared using reagents supplied to each kit, applying the conditions for formulation of the reagents recommended by the kit, in 10 μl of a reaction system. However, as a reverse transcription primer, an oligo (dT) 18 primer was used in all of the kits. 1.5 μl of RNA-primer mix (20 ng of Millennium RNA Markers, 25 pmol of oligo (dT) 18 primer, and lysis buffer (RamDA-A conditions)) was denatured at 65° C. for 2 minutes, it was then mixed with 8.5 μl of reverse transcription mix, and the obtained mixture was then reacted at 42° C. for 2.5 minutes, and then at 85° C. for 5 minutes. After completion of the reaction, the reaction mixture was analyzed by agarose gel electrophoresis.
For an adaptation test of the RamDA method, using 10 pg of total RNA derived from mouse ES cells, a reverse transcription reaction was carried out. For the preparation of a reverse transcription mixture, reagents supplied to each kit were used, and for formulation of the reagents, 3 μl of a reaction system, which was down-scaled under conditions recommended by the kit, was used. However, regarding reverse transcription primers, 0.6 pmol of oligo (dT) 18 primer and 7.8 pmol of random hexamer primer were used under RamDA (−) conditions, whereas 0.6 pmol of oligo (dT) 18 primer and 7.8 pmol of ZNA-random hexamer primer were used under RamDA (+) conditions. Moreover, in the case of RamDA (+), 0.2 U of dsDNase (ArcticZymes) and 100 ng of T4 gene 32 protein were further added. The reaction was carried out under reaction temperature conditions of 25° C., 10 minutes, 30° C., 10 minutes, 42° C., 30 minutes, 50° C., 5 minutes, and 85° C., 5 minutes, and the amplification rate of cDNA was then quantified by qPCR.
Preparation of reagents and the reactions were all carried out on 0.2 ml of Hi-Tube Flat Cap Recovery (TaKaRa), EU Semi-domed 8-cap strip (BIOplastics BV, Landgraaf, Netherlands), or RP, LF, SEMI Sk, cutable, 96 well plate (BIOplastics BV).
Quantitative Polymerase Chain Reaction (qPCR)
In the case of an artificially synthesized RNA (Dap and Thr) system, a reverse transcription reaction product was diluted with nuclease free water (Qiagen), and the diluted product in an amount of 1/50 was used as a qPCR reaction solution. In the case of a system of 10 pg of Total RNA derived from mouse ES cells, a reverse transcription reaction product was diluted with nuclease free water, and the diluted product in an amount of ⅙, ⅛, or 1/10, was used as a qPCR reaction solution. The qPCR was carried out using LightCyclar 480 (Roche) or ABI 7900HT (Life Technologies) under the following conditions. 10 μl of qPCR reaction solution (1× Quantitect SybrGreen master mix (Qiagen), 5 pmol of forward primer, 5 pmol of reverse primer, 3 μl of diluted cDNA, and nuclease free water) was reacted at 95° C. for 15 minutes to activate the enzyme, and thereafter, denaturation at 95° C. for 15 seconds and an elongation reaction at 60° C. for 1 minute were carried out for 40 cycles. The melting curve was analyzed at 95° C. for 15 seconds, at 60° C. for 15 seconds, and at 95° C. for 15 seconds. For the production of standard curves used for absolute quantification, as standard DNAs, 5-fold dilution series of a dsDNA mixed solution of Dap and Thr (31250, 6250, 1250, 250, 50, 10, and 0 copy) were used for artificially synthesized RNA, and 5-fold dilution series of mouse genomic DNA (Clonetech) (31250, 6250, 1250, 250, 50, 10, and 0 copy) were used for total RNA. Since cDNA was ssDNA, the quantitative value was calculated by the formula: a detected value (dsDNA copy)×2. The sequences of individual primers are shown in Table 2. Data analysis was carried out using LightCycler 480 Software, Version 1.5 (Roche) or SDS Software 2.1 (Life Technologies).
Gel Electrophoresis
Each reverse transcription reaction product was diluted with an RNase-free TE buffer (Life Technologies) to result in a volume of 20 μl, and the thus diluted product was subjected to a denaturation treatment at 70° C. for 10 minutes, and immediately after the treatment, the reaction solution was cooled on ice. Regarding DNA ladder markers, 1 μl of E-Gel 1 Kb Plus DNA Ladder (Life Technologies), which was diluted with an RNase-free TE buffer to a volume of 20 μl and was then thermally denatured, was used as an ssDNA ladder, and 1 μl of E-Gel 1 Kb Plus DNA Ladder (Life Technologies) which was diluted but was not thermally denatured, was used as a dsDNA ladder. However, in a system of using Millennium RNA Markers, a thermal denaturation treatment was omitted after completion of the dilution. The reverse transcription reaction product and the ladder marker were filled into 2% E-Gel EX Agarose Gels (Life Technologies), and thereafter, electrophoresis was carried out using E-Gel iBase Power System (Life Technologies) for 10 minutes. Electrophoretic images were photographed using FAS-Digi (NIPPON Genetics Co. Ltd, Tokyo, Japan).
(Results)
Selective Cleavage of DNA in RNA-DNA Hybrid by dsDNase and DNase I
First, whether dsDNase or DNase I has an activity of selectively cleaving DNA in an RNA-DNA hybrid under standard reserve transcription buffer conditions was examined.
Crab-derived double strand-specific nuclease has been known to have an activity of cleaving DNA in an RNA-DNA hybrid (Non Patent Literature 11). However, this enzyme has almost no activity at a temperature applied during reverse transcription (Non Patent Literature 11). As such, shrimp-derived dsDNase known as a thermally unstable enzyme has been focused (Non Patent Literature 12). This enzyme has an activity around 37° C. and specifically cleaves double-stranded DNA. Thus, this enzyme has an action to remove contaminated DNA, genomic DNA and the like without digesting primers or cDNA (Non Patent Literatures 12 and 13). However, it had not yet been reported that this enzyme has an activity of cleaving DNA in an RNA-DNA hybrid, which is essential for carrying out a strand displacement reaction. Hence, using Fluorescence resonance energy transfer (FRET), an analysis was carried out. As a result, it was found that dsDNase has an activity of selectively cleaving the DNA in an RNA-DNA hybrid, although the activity is about a half of the activity on double-stranded DNA (
Amplification by Fragmentation of cDNA and Strand Displacement Reaction
In order to confirm whether dsDNase actually acts on cDNA synthesized by a reverse transcription reaction so as to fragment it, and also, whether a strand displacement reaction thereby takes place and cDNA is amplified, reverse transcription was carried out using about 2 kb of poly-A-added artificially synthesized RNA as a template, and thereafter, the presence or absence of fragmentation of cDNA was examined by gel electrophoresis, and the yield of cDNA was measured by RT-qPCR. Consequently, it was found that, in a standard reverse transcription method (RamDA (−)), almost no fragmentation took place in both cases of an oligo dT primer and a random hexamer primer. On the other hand, in RamDA (+), cDNA was fragmented, and a smear electrophoretic image was obtained (
dsDNase Enzyme and Reverse Transcriptase that can be Used in RT-RamDA Method
In order to examine a dsDNase enzyme and reverse transcriptase that can be used in an RT-RamDA method, whether amplification effects can be obtained even under conditions of using other dsDNase enzymes or various types of reverse transcriptases was examined. First, regarding dsDNase enzymes, a comparison was made among three enzymes, namely, dsDNase (43 KDa) commercially available from ArcticZymes, dsDNase (47 KDa) of Affymetrix, and duplex specific nuclease (DSN) of Evrogen. Using 10 pg of total RNA derived from mouse ES cells that was the amount of total RNA corresponding to a single cell, reverse transcription was carried out. As a result, a significant increase in the detected amount of cDNA was observed in the two dsDNase-added samples (
Next, the presence or absence of amplification effects obtained by the RT-RamDA method was examined using commercially available reverse transcriptase. First, to examine the elongation activity of an enzyme, poly-A-added Millennium RNA Markers (Life Technologies) were used to perform reverse transcription using an oligo dT primer, so that a performance test was carried out. As a result, it was found that, regardless of a short elongation time that was 2 minutes 30 seconds, almost all enzymes had an elongation activity of 2 kb or more (
Next, individual reverse transcriptases were used to perform reverse transcription using 10 pg of total RNA derived from mouse ES cells, under conditions of addition (+) of RT-RamDA components (dsDNase, a T4 gene 32 protein, and a ZNA-random hexamer primer), or non-addition (−) of such components, and a comparison was then made by qPCR, in terms of the yield of cDNA. As a result, in all of reverse transcriptases having no RNase H activity, in the case of RamDA (+), an increase in the detected amount was observed (
Concerning Method of Enhancing Reaction Efficiency of RT-RamDA Method
The present inventors have focused on the improvement of an amplification rate, and have developed deviation methods, namely, an RT-RamDA method (RamDA-B) without using a T4 gene 32 protein and an RT-RamDA thermal cycling method (RamDA-C). In a strand displacement reaction, formation of the secondary structure of a template hinders amplification. The present inventors have introduced a T4 gene 32 protein into the RT-RamDA method (RamDA-A) as a role for alleviating such a secondary structure. However, at the same time, it was assumed that the T4 gene 32 protein would cause instabilization of a DNA-RNA hybrid, and would suppress the activity of dsDNase. As such, RamDA-B without using such a T4 gene 32 protein was attempted. Using 10 pg of total RNA derived from mouse ES cells as a template, the synthesis of cDNA was carried out. As a result, it was found that, under the conditions of RamDA-B, the detected amounts of Gnb2l1 and Oct3/4 were increased to 40 times or more, in comparison to a standard reverse transcription method (RamDA (−)) (
Next, RamDA-C, in which only the reaction temperature conditions in RamDA-B were modified and an annealing step and an elongation were repeated with short intervals, was attempted. As a result, it was found that the detected amounts of Gnb2l1 and Oct3/4 in RamDA-C were increased to 100 times or more those in a standard reverse transcription method (
Concerning RT-RamDA Method of Using DNase I
The greatest reason for the use of dsDNase in the RT-RamDA method is that the dsDNase does not have a nuclease activity on amplified cDNA that is single-stranded DNA, and a reverse transcription primer. However, it has been known that DNase I that is a non-specific DNA degrading enzyme originally has a lower nuclease activity on single-stranded DNA, than on double-stranded DNA (Non Patent Literature 12), and that its nuclease activity is reduced in a monovalent cation (e.g. K+, Na+, etc.) dependent manner (Non Patent Literatures 23 and 24). On the other hand, the general composition of a reaction buffer used in reverse transcription (First-Strand buffer (Life Technologies), PrimeScript Buffer (for cDNA synthesis) (TaKaRa), Maxima H Minus First Strand cDNA Synthesis RT-Buffer (Thermo Fisher), M-MuLV Reverse Transcriptase Reaction Buffer (New England Biolabs), AffinityScript RT Buffer (Agilent Biotechnology), etc.) comprises 50 mM Tris-HCl, 75 mM KCl, and 3 mM MgCl2. Thus, the concentrations of Tris-HCl and KCl are high in such a reaction buffer used in reverse transcription. That is, it was predicted that the nuclease activity of DNase I on single-stranded DNA would be low under the conditions of the reverse transcription reaction buffer, and the RT-RamDA method would function. In reality, according to FRET analysis, nuclease activity was measured on the basis of 20 mM Tris-HCl, 50 mM KCl and 2 mM MgCl comprised in DNase I reaction buffer (Life Technologies), by increasing the concentrations of Tris-HCl and KCl. As a result, it was found that the nuclease activity of DNase I, not only on double-stranded DNA, but also on single-stranded DNA and DNA in an RNA-DNA hybrid, is significantly inhibited under conditions of high concentrations of Tris-HCl and KCl (
Next, the efficacy of the RT-RamDA method of using a non-specific DNA degrading enzyme, and in particular, the relationship between the salt concentration in the reaction solution and an amplification rate was examined. The salt concentration was changed from the composition of a DNase I reaction buffer to the composition of a reverse transcription reaction buffer, and whether the RT-RamDA method functions was examined using qPCR. As a result, unexpectedly, it was found that the RT-RamDA method provides sufficient amplification even under conditions of a DNase I reaction buffer having a high nuclease activity on single-stranded DNA (
Contribution of T4 Gene 32 Protein to Protection of cDNA and Stabilization of Amplification
It has been reported that the T4 gene 32 protein has an action to bind to single-stranded DNA, so as to protect the single-stranded DNA from nuclease (Non Patent Literature 25). Thus, the fragmentation state of cDNA according to the RT-RamDA method of using, as a template, poly-A-added artificially synthesized RNA, and also using only an oligo dT primer, was examined based on the presence or absence of a T4 gene 32 protein (
T4 Gene 32 Protein Having Action to Improve Ratio Between Nuclease Activity on DNA in RNA-DNA Hybrid Strand and Activity on Single-Stranded DNA
For the function of the RT-RamDA method of using DNase I, it is considered important that the decomposed amount of amplified DNA is smaller than the amount of strand displacement amplification caused by formation of a nick in an RNA-cDNA strand, namely, that nuclease activity on single-stranded DNA is sufficiently smaller than activity on DNA in an RNA-DNA hybrid strand. From an experiment regarding fragmentation of cDNA and amplification rate (
RamDA-D Showing Reaction Time-Dependent Amplification Rate
Whether the amplification rate would be improved in a reaction time-dependent manner was examined using a 1-cell lysate of mouse ES cells as a template (
RamDA-D that is not Limited to Specific RNase H Minus Reverse Transcriptase, and in Addition, Functions Also on RNA Corresponding to 100 Cells.
In order to confirm whether RamDA-D is limited to a specific RNase H minus reverse transcriptase, an examination was carried out using reverse transcriptase of SuperScript series (Life Technologies), as well as PrimeScript RT Enzyme Mix I (
As stated above, the RT-RamDA method can be an extremely useful means as a technique of amplifying trace RNA, and in particular, the amplification rate provided by RamDA-C should be an extremely great advantage in the analysis of detecting a specific target gene. On the other hand, since RamDA-C causes a large fluctuation in the amplification rates among genes, RamDA-A involving the use of a T4 gene 32 protein is effective for an analysis requiring uniform amplification. Moreover, RamDA-D involving the use of such a T4 gene 32 protein and DNase I as a non-specific DNA degrading enzyme has a high amplification rate and also has a small amplification fluctuation among genes or within a single gene. Furthermore, since the nuclease activity of DNase I on double-stranded DNA during a reverse transcription reaction is also maintained at a level higher than that of a double strand-specific DNA degrading enzyme, it is expected that RamDA-D will have the effect of reducing the influence of DNA contamination. As described above, RamDA-D is a method comprising both the superiority of RamDA-A and that of RamDA-B, and this is an extremely effective and simple reverse transcription method targeting a trace amount of RNA.
The present application claims priority from Japanese Patent Application No. 2014-200258 (filed on Sep. 30, 2014); the disclosure of which is hereby incorporated by reference in its entirety. In addition, all patent publications, patent applications, and publications cited herein are incorporated herein by reference in their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2014-200258 | Sep 2014 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2015/077745 | 9/30/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/052619 | 4/7/2016 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20050084875 | Coleman et al. | Apr 2005 | A1 |
| 20110020878 | Lanes et al. | Jan 2011 | A1 |
| 20140213485 | Weissman | Jul 2014 | A1 |
| Number | Date | Country |
|---|---|---|
| 1707623 | Oct 2006 | EP |
| 1736775 | Dec 2006 | EP |
| 2666870 | Nov 2013 | EP |
| 2014-103867 | Jun 2014 | JP |
| 2014103867 | Jun 2014 | JP |
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| Number | Date | Country | |
|---|---|---|---|
| 20170275685 A1 | Sep 2017 | US |
