JP2011147415A - Method for uniform amplification of nucleic acid and highly sensitive detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for rapidly and readily amplifying a small amount of nucleic acids in high amplification factor. <P>SOLUTION: The method for amplification of the nucleic acids includes a step for introducing a repeated sequence containing an already-known sequence to the 3' terminus of a target nucleic acid chain containing a target sequence or a complementary sequence thereof, a step for hybridizing a plurality of oligonucleotides hybridizable with the repeated sequence, with the repeated sequence introduced into the target nucleic acid chain, and a step for carrying out the synthesis of a complementary chain using the target nucleic acid chain as a template by using the oligonucleotide as a primer to form a plurality of the nucleic acid-complementary chains from a single target nucleic acid chain. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a nucleic acid amplification method and a detection method. Specifically, the present invention relates to a method for amplifying DNA or mRNA uniformly and efficiently, and a method for detecting a nucleic acid using the amplification method.

  In comparative analysis of various DNA (or mRNA) such as gene expression analysis and gene diagnosis, since the copy number of the target DNA is small, it is widely performed after the copy number is amplified. In general amplification methods, PCR is performed by increasing the number of copies by selecting two sites as a priming region across the target DNA region, hybridizing the primer to that region, and repeatedly synthesizing the target region with complementary strands. Polymerase Chain Reaction) or hybridizing a probe to the target region, ligation produces a ring-shaped DNA, and complementary strand synthesis is performed over the circumference of the ring using primers that hybridize to it. RCA (Rolling Circle Amplification) that amplifies the region or strand displacement amplification (SDA) is known. In addition, a DNA amplification method combining many primer sets and strand displacement amplification has been reported (Patent Document 1). However, in these amplification methods, the amplification factor may vary depending on the length of the region to be amplified and the sequence or type of the constituent bases. This is a major problem in examining detailed gene expression.

  Gene expression analysis is not only important as basic data in life science, but is also used in a wide range of fields such as diagnosis and drug discovery. Currently, many cells are used as samples to investigate gene expression, mRNA is taken out to produce cDNA, and these are collectively amplified by PCR, etc., and then expressed using a DNA chip or the like. Quantitative analysis. Not only the measurement using these many cells but also the analysis of the gene expressed in one cell is drawing attention (Non-patent Document 1). Such gene expression analysis requires precise quantitative analysis using much less mRNA than the method using many cells. For this reason, amplification of mRNA is important. Currently, a reagent kit for performing batch amplification on mRNA is commercially available (QIAGEN; QuantiTect Whole Transcriptome Kit). This is because when cDNA is generated from mRNA, cDNA is prepared using random primers that hybridize to various sites, and the resulting cDNA fragment mixture is ligated to form a ring-shaped DNA in which various fragments are mixed. Then, RCA amplification is performed by reacting the random primer again. This method works relatively well when many cells of 500 to 1000 cells or more are used, but the amplification rate for each target DNA fragment changes with a small number of cells. In addition, a collective amplification method using PCR is also used (Non-patent Document 1). First, a cDNA is prepared from mRNA using a DNA probe having a poly-T sequence with an anchor sequence, and its 3 ′ end. A terminal transferase is used to add a poly A chain to form a template DNA for amplification. PCR amplification is performed using the anchor sequence and the poly A sequence portion, but the amplification rate also varies depending on the sequence and length of the target. A uniform DNA amplification method is desired for more precise gene expression analysis.

When performing precise comparative analysis such as gene expression analysis using a sample containing a small number of DNA copies, it is indispensable to uniformly amplify various target DNAs. The polymerase chain reaction (PCR) method generally used as an amplification method is a method of repeating a process of synthesizing complementary strands by raising and lowering the temperature to make double-stranded DNA into a single strand and hybridizing primers. The efficiency of complementary strand synthesis varies slightly depending on the DNA sequence and length. In PCR, amplification is about Y ⁿ times when Y is the amplification factor per cycle and n is the number of temperature cycles. Y is ideally 2 but actually around 1.6. n is usually 30 to 40, but if the amplification factor per thermal cycle changes slightly, the amplification factor changes greatly. For example, if Y is changed by 2% and the heat cycle reaction is performed 35 times, the amplification factor can be doubled. Since the amplification rate per thermal cycle depends on the temperature and the length and sequence of the target DNA, a difference in amplification rate of several times can be made when the DNA type is different. Such a difference is an obstacle when measuring the precise abundance of DNA.

  On the other hand, in the rolling circle amplification (RCA) method in which ring-shaped DNA is prepared and primers are hybridized and complementary strand synthesis is repeated, the amplification rate changes depending on the size of the target ring, but the complementary strand synthesis time There is an advantage that the amplification factor is almost determined. However, it is necessary to make the region to be amplified ring-shaped DNA, which is effective when amplifying short known sequence parts, but for amplification of long unknown sequence parts, amplification per unit time The rate is low and unsuitable. For example, when trying to amplify cDNA obtained from mRNA for gene expression profile analysis, RCA cannot be used because the lengths of the cDNAs differ.

  In addition, the method using multiple primer sets and strand-displacing DNA complementary strand synthesis requires many primer sets for each target DNA, so it is complicated when there are multiple target DNA fragments. Unexpected by-products may be generated and are still not effective.

  Furthermore, genetic diagnosis using DNA is used for the determination of infectious diseases, and it is desired to amplify and detect DNA as quickly as possible, but at present it takes at least about 1 hour. For example, in PCR, the time required for one thermal process is usually about 1 to 2 minutes. A full amplification requires about 40 thermal cycles, which takes an hour. Such a DNA amplification test requiring a long time is not desirable for testing for infectious diseases such as viruses. This is because the inspection is performed in the shortest possible time and the subsequent policy is determined. Therefore, a method is desired which has a large amplification factor and can amplify DNA in a short time.

JP-T-2001-519172

Nucreic Acids Res. 34, e42 (2006)

  As described above, there is a need to establish a method for amplifying DNA in a short time, which can be applied to a small amount of DNA amplification, regardless of the sequence or length of DNA.

  Accordingly, an object of the present invention is to provide a method for amplifying a small amount of nucleic acid quickly and easily at a high amplification factor. Another object of the present invention is to provide a method for rapidly amplifying and detecting a small amount of nucleic acid at a high amplification rate.

  As a result of intensive studies to solve the above problems, the present inventor introduced a repetitive sequence at the end of the nucleic acid to be amplified and hybridized a plurality of primers to the repetitive sequence to synthesize a strand displacement type complementary strand. As a result, it was possible to synthesize the nucleic acid to be amplified and its complementary strand simultaneously in parallel, and obtained the knowledge that the nucleic acid could be amplified at a high amplification rate in a short time, and the present invention was completed.

  That is, the present invention is as follows.

(1) A method for amplifying nucleic acid,
Introducing a repetitive sequence containing a known sequence at the 3 ′ end of the target nucleic acid strand containing the target sequence or its complementary sequence;
Hybridizing a plurality of oligonucleotides that hybridize to the repetitive sequence to the repetitive sequence introduced into the target nucleic acid chain;
A method comprising a step of synthesizing complementary strands using the target nucleic acid strand as a template using the oligonucleotide as a primer to generate a plurality of nucleic acid complementary strands from one target nucleic acid strand.
(2) The method according to (1), wherein the repetitive sequence is shorter than the length of the target sequence or its complementary sequence.
(3) The method according to (1), wherein the repetitive sequence is longer than the length of the target sequence or its complementary sequence.
(4) The method according to any one of (1) to (3), wherein the number of repeating units contained in the repeating sequence exceeds 100.
(5) The method according to any one of (1) to (3), wherein the number of repeating units contained in the repeating sequence exceeds 1000.
(6) The target nucleic acid strand includes a plurality of types of nucleic acid strands, and a common repetitive sequence is introduced into the plurality of types of nucleic acid strands. (1) to (5) The method described.

(7) The method according to any one of (1) to (6), wherein the introduction of the repeating sequence is performed by introducing a nucleic acid chain having the repeating sequence into the target nucleic acid chain by ligation.
(8) Introducing a repetitive sequence, using a nucleic acid chain having a repetitive sequence as a template, repetitively sequencing the 3 'end portion of the target nucleic acid strand as a repetitive sequence introduction primer or a sequence portion added to the 3' end of the target nucleic acid strand The method according to any one of (1) to (6), which is performed by synthesis of a complementary strand used as a primer for introduction.
(9) The method according to (7) or (8), wherein the nucleic acid strand having a repetitive sequence is produced by complementary strand synthesis using a ring-shaped nucleic acid as a template.

(10) The introduction of a repetitive sequence is performed by complementary strand synthesis using a sequence portion contained in the target nucleic acid strand or a sequence portion added to the target nucleic acid strand as a primer for repetitive sequence introduction and using a ring-shaped nucleic acid as a template. The method according to any one of (1) to (6).
(11) An oligonucleotide capable of forming a ring-shaped nucleic acid by hybridizing to the sequence portion by using the sequence portion added to the target nucleic acid strand as a repeat sequence-introducing primer for introduction of the repeated sequence and ligation The method according to any one of (1) to (6), wherein the method is performed by complementary strand synthesis using a ring-shaped nucleic acid formed by using as a template.
(12) The method according to (10) or (11), wherein at least part of the primer for introducing a repeated sequence uses a sequence portion added to the 3 ′ end of the target nucleic acid strand.
(13) Repeated sequence introduction introduces the complementary sequence of the sequence portion obtained by complementary strand synthesis using an oligonucleotide that hybridizes to at least part of the sequence portion added to the 3 'end of the target nucleic acid strand as a template. The method according to any one of (1) to (6), wherein the method is carried out by complementary strand synthesis using a ring-shaped nucleic acid as a template.
(14) The method according to any one of (8) to (13), wherein a sequence portion is added to the 3 ′ end of the target nucleic acid strand by terminal transferase.
(15) Hybridizes to a sequence portion on the 3 ′ end side of the target nucleic acid strand adjacent to the sequence portion added to the 3 ′ end of the target nucleic acid strand, and all or part of the added sequence portion, and a known sequence Complementary strand synthesis is performed using the contained oligonucleotide as a template, a part of the sequence portion added to the 3 ′ end of the target nucleic acid strand is removed as necessary, and then the hybridized oligonucleotide is used as a template for complementary strand synthesis. Using the known sequence added to the 3 ′ end of the target nucleic acid strand as a primer for repetitive sequence introduction, a repetitive sequence is introduced by carrying out complementary strand synthesis using a ring nucleic acid as a template. The method described in 13).
(16) Removal of a part of the sequence portion added to the 3 ′ end of the target nucleic acid strand is achieved by removing the base of the sequence portion where the oligonucleotide is not hybridized using an enzyme containing exonuclease. The method according to (15), which is performed.

(17) The sequence portion used as a primer for repetitive sequence introduction added to the 3 'end of the target nucleic acid strand is generated by oligonucleotide ligation following restriction enzyme cleavage or complementary strand synthesis using the oligonucleotide as a template. The method according to any one of (10) to (13), wherein
(18) The method according to (10), wherein a known sequence portion contained in the target nucleic acid chain is used as a primer for introducing a repeated sequence.
(19) The method according to (18), wherein a known sequence portion of the target nucleic acid chain is cleaved, and a portion including the 3 ′ end of the obtained target nucleic acid strand is repeatedly used as a primer for sequence introduction.
(20) The method according to (19), wherein the known sequence portion of the target nucleic acid strand is cleaved with an enzyme containing Cleavase.

(21) Two strands are synthesized by hybridizing to a known sequence portion of the target nucleic acid strand and hybridizing an oligonucleotide having an anchor sequence on the 5 ′ end side to the target nucleic acid strand and extending the oligonucleotide. A single-stranded portion of the target nucleic acid strand is decomposed and removed using an enzyme that generates a single-stranded nucleic acid and degrades the single-stranded nucleic acid from the 3 ′ end, and then 3 ′ of the target nucleic acid strand using the oligonucleotide anchor sequence as a template. The method according to any one of (10) to (13), wherein a sequence portion is added to a target nucleic acid strand by complementary strand synthesis extending on the terminal side to generate a primer for repeated sequence introduction.
(22) Two strands are synthesized by hybridizing to a known sequence portion of the target nucleic acid strand and hybridizing to the target nucleic acid strand an oligonucleotide having an anchor sequence on the 5 ′ end side and extending the oligonucleotide. A strand nucleic acid is generated, a single strand portion of the target nucleic acid strand is removed, and then a known sequence portion is added to the 3 ′ end of the target nucleic acid strand by complementary strand synthesis using the anchor sequence of the oligonucleotide as a template. The method according to any one of (10) to (13), wherein a primer for repetitive sequence introduction is generated by removing nucleotides.
(23) A known sequence portion is added to the 3 ′ end and 5 ′ end of the target nucleic acid strand, and the known sequence portion at the 3 ′ end of both the target nucleic acid strand and its complementary nucleic acid strand is used as a primer for repetitive sequence introduction. (8) to (8), wherein a cyclic sequence is introduced into the 3 ′ end and the 5 ′ end by synthesizing a complementary strand using a ring-shaped DNA capable of hybridizing to a known sequence portion as a template. The method according to any one of 11).
(24) Synthesizing a complementary strand using as a primer the first oligonucleotide that hybridizes to a repetitive sequence introduced into the target nucleic acid strand, and using the generated complementary nucleic acid strand as a template to hybridize to the 3 ′ end. The method according to any one of (1) to (23), comprising a step of performing complementary strand synthesis using the oligonucleotide of 2 as a primer and amplifying a copy of the target nucleic acid strand.
(25) Performing complementary strand synthesis using an oligonucleotide that hybridizes to the repetitive sequence introduced into the target nucleic acid strand as a primer, the resulting double-stranded nucleic acid as a single-stranded nucleic acid, and then using the oligonucleotide as a primer, The method according to any one of (1) to (23), wherein the step of performing complementary strand synthesis using the generated single-stranded nucleic acid as a template is repeated.

(26) The method includes any one of (1) to (25), including a step of generating a target nucleic acid strand containing a target sequence or a complementary sequence thereof, having a repeat sequence at the 3 ′ end, and having a loop sequence at the 5 ′ end The method according to
(27) The method according to any one of (1) to (26), wherein the nucleic acid strand is a DNA strand.

(28) The method according to any one of (1) to (27), wherein the 5 ′ end side of the target nucleic acid strand is immobilized on a solid surface.
(29) The method according to any one of (1) to (28), wherein an oligonucleotide that hybridizes to a repetitive sequence introduced into the target nucleic acid chain is immobilized on a solid surface.
(30) A complementary nucleic acid chain generated using the first oligonucleotide that hybridizes to the repetitive sequence introduced into the target nucleic acid chain is captured using the second oligonucleotide immobilized on the solid surface, and the second The method according to any one of (1) to (29), comprising a step of performing complementary strand synthesis using the oligonucleotide of (1) as a primer.
(31) The method according to any one of (28) to (30), wherein the solid is a bead.
(32) The method according to any one of (28) to (30), wherein the solid is a partitioned planar solid.
(33) The method according to any one of (28) to (30), wherein the solid surface is a porous or inner wall of a pore array.
(34) The method according to any one of (28) to (33), wherein at least two kinds of oligonucleotides that hybridize to the target nucleic acid strand or its complementary nucleic acid strand are immobilized on the same solid surface.

(35) Perform complementary strand synthesis so that a restriction enzyme cleavage recognition sequence is introduced before or after the repetitive sequence introduced into the target nucleic acid strand, and cleave the repetitive sequence from the obtained complementary nucleic acid strand using the restriction enzyme. The method according to any one of (1) to (34), comprising a removing step.
(36) a step of synthesizing a complementary strand using a target nucleic acid strand as a template using a capture probe having a sequence complementary to the target nucleic acid strand and a first anchor sequence at the 5 ′ end; A known sequence based on the first anchor sequence on the 3 ′ end side where the first oligonucleotide is extended by synthesizing the complementary strand using the first oligonucleotide hybridizing to the single-stranded complementary nucleic acid strand A step of introducing a portion, a step of hybridizing to a ring-shaped nucleic acid strand using the known sequence portion as a primer to synthesize a complementary strand, and generating a complementary nucleic acid strand having the first repetitive sequence introduced at the 3 ′ end side And hybridizing a plurality of second oligonucleotides to the first repetitive sequence and synthesizing complementary strands in parallel, (1) The method described in 1.
(37) The first oligonucleotide contains the second anchor sequence on the 5 ′ end side, and the 3 ′ end side of the complementary strand synthesized with the second oligonucleotide hybridizing to the first repeat sequence as a primer. A known sequence portion based on the second anchor sequence is introduced into the base, and the known sequence portion is used as a primer to synthesize a complementary strand using a ring-shaped nucleic acid as a template. The method according to (36), further comprising the step of generating an introduced complementary nucleic acid strand.
(38) A cDNA library is prepared from a plurality of types of mRNA, and the DNA is uniformly amplified using a plurality of types of cDNAs contained in the cDNA library as a target nucleic acid chain, according to any one of (1) to (37) the method of.
(39) The method according to (38), wherein an amplified DNA copy is produced on a bead different from the bead that captures mRNA or cDNA.

(40) A method for uniformly amplifying a nucleic acid pool containing single or multiple nucleic acid strands,
A method comprising a step of introducing a repetitive sequence containing a known sequence at the 3 ′ end of a nucleic acid strand contained in a nucleic acid pool, an oligonucleotide hybridizing to the repetitive sequence, and a step of performing complementary strand synthesis using a strand displacement polymerase .
(41) A method for amplifying a nucleic acid, the step of introducing a repetitive sequence containing a known sequence at the 3 ′ end of a target nucleic acid strand containing a target sequence or its complementary sequence, using an oligonucleotide that hybridizes to the repetitive sequence A method comprising a step of performing complementary strand synthesis, a step of introducing a cleaved portion (nick) into the generated double-stranded nucleic acid, and a step of performing complementary strand synthesis from the cleaved portion (nick) to generate a plurality of nucleic acid strands.
(42) A method for detecting a nucleic acid in a sample, the step of amplifying the nucleic acid in the sample by the method according to any one of (1) to (41), and using the obtained amplification product, Detecting the nucleic acid.
(43) The method according to (42), wherein all the nucleic acids in the sample or only the nucleic acids to be detected are amplified.

  The present invention provides a nucleic acid amplification method. Such a method can amplify any nucleic acid at the same amplification factor even if plural kinds of nucleic acids coexist. In addition, according to such a method, it is possible to amplify a nucleic acid uniformly at a high amplification rate in a short time regardless of the sequence or length of the nucleic acid. Therefore, the amplification method according to the present invention is excellent as a method for uniformly amplifying mRNA, in particular, synthesizing cDNA from a small amount of mRNA obtained from one cell and amplifying the cDNA uniformly without bias. The method according to the present invention is useful in the fields of gene expression analysis, cell function analysis, biological tissue analysis method, disease diagnosis, infectious disease test, drug discovery, etc., where rapid nucleic acid amplification and detection are desired. .

1 shows an overview of one embodiment of the present invention. A known sequence is introduced into the 3 ′ end of the target DNA, and this strand is used as a primer (primer P1-1) to synthesize a complementary strand using the ring DNA as a template, and a repeated sequence is introduced into the 3 ′ end of the target DNA. To do. Next, complementary strand synthesis is performed in parallel using a primer (primer P1-2) that hybridizes to the repetitive sequence portion, and a complementary strand copy of the target DNA is amplified. Next, complementary strand synthesis is performed to amplify a DNA strand having a homologous sequence with the target DNA. It is a continuation of FIG. 1-1a. It is a continuation of FIG. 1-1a. The structure of ring DNA 1 used for the amplification reaction is shown. As an embodiment of the present invention, an outline is shown in which a known sequence portion is added by a method using loop probe hybridization and the enzyme Cleavase. The structure of an example of ring DNA used for amplification reaction is shown. As one embodiment of the present invention, the principle of a method for amplifying DNA with high efficiency by introducing repetitive sequences at both ends is shown. It is a continuation of FIG. As an embodiment of the present invention, an outline of a method for introducing a repetitive sequence into the end of a target DNA using a capture probe fixed to the bead using an example in which the target DNA is fixed to the bead will be described. It is a continuation of FIG. 3-2a. As an embodiment of the present invention, an outline of a method for amplifying by introducing a loop sequence and a repetitive sequence into a target DNA is shown. It is a continuation of FIG. As an embodiment of the present invention, an outline of a method using a bead-immobilized target DNA and an amplification primer immobilized on a solid surface is shown. Numerous amplification products can be generated from a single DNA in a narrow area of the solid surface. It is a continuation of FIG. As an embodiment of the present invention, examples of various methods for immobilizing a probe are shown. As an embodiment of the present invention, an outline of a method for synthesizing a complementary strand from a nick by introducing an enzyme recognition site that generates a cleavage site (nick) during complementary strand synthesis will be described. It is a continuation of FIG.

The present invention relates to a method for amplifying nucleic acids uniformly and efficiently. Specifically, the nucleic acid amplification method of the present invention comprises:
Introducing a repetitive sequence containing a known sequence at the 3 ′ end of the target nucleic acid strand containing the target sequence or its complementary sequence;
Hybridizing a plurality of oligonucleotides that hybridize to the repetitive sequence to the repetitive sequence introduced into the target nucleic acid chain;
Using the oligonucleotide as a primer to synthesize a complementary strand using the target nucleic acid strand as a template, and generating a plurality of nucleic acid complementary strands from one target nucleic acid strand.

  The target nucleic acid strand containing the target sequence or its complementary sequence is not particularly limited as long as it contains the sequence to be amplified. Deoxyribonucleic acid (DNA) such as cDNA and ribonucleic acid (RNA) such as messenger RNA (mRNA), and fragments and hybrid nucleic acids thereof. The target nucleic acid strand can be prepared by a method known in the art. For example, when preparing a target nucleic acid from cells, use a proteolytic enzyme such as Proteinase K, a chaotropic salt such as guanidine thiocyanate or guanidine hydrochloride, a surfactant such as Tween or SDS, or a commercially available reagent for cell lysis. The cells can be lysed and the nucleic acids contained therein, ie DNA and RNA, can be eluted. When RNA is prepared, among the nucleic acids eluted by cell lysis, DNA is decomposed with a DNA degrading enzyme (DNase), and a sample containing only RNA as a nucleic acid is obtained. When mRNA is prepared, since the mRNA contains a poly A sequence, only the mRNA can be captured from the RNA sample prepared as described above using a DNA probe containing a poly T sequence. When preparing cDNA, cDNA can be synthesized from the mRNA obtained as described above by performing a reverse transcription reaction using a reverse transcriptase. For example, a DNA containing a poly-T sequence used at the time of mRNA preparation. A cDNA can be synthesized by reacting with a reverse transcriptase in the presence of deoxynucleotide using a probe as a primer and mRNA as a template.

  The target nucleic acid strand may include a single type of nucleic acid strand, or may include a plurality of types of nucleic acid strands. That is, the target nucleic acid strand may include the same target sequence or a complementary sequence thereof, or may include different sequences. For example, the target nucleic acid strand can be a nucleic acid pool, a cDNA library, or the like. For example, in the present invention, a cDNA library can be prepared from a plurality of types of mRNA, and the plurality of types of cDNAs contained in the cDNA library can be uniformly amplified as target nucleic acid chains.

  Further, the target nucleic acid chain may be in a free state, or the 5 ′ end side may be fixed on the solid surface. The solid to which the target nucleic acid chain can be immobilized is not particularly limited as long as it is a solid generally used for nucleic acid manipulation. Specifically, it is preferably a solid that is insoluble in water and does not melt during heat denaturation. Examples of the material include metals such as gold, silver, copper, aluminum, tungsten, molybdenum, chromium, platinum, titanium, and nickel; alloys such as stainless steel, hastelloy, inconel, monel, and duralumin; silicon; glass, quartz glass, Glass materials such as fused quartz, synthetic quartz, alumina, sapphire, ceramics, forsterite and photosensitive glass; polyester resin, polystyrene, polyethylene resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), nylon, acrylic resin, fluorine resin , Polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin and other plastics; agarose, dextran, cellulose, polyvinyl alcohol, nito Cellulose, chitin, chitosan. Further, the solid shape is not particularly limited, and examples thereof include compartmentalized flat surfaces (for example, titer plates, porous or fine pore arrays), flat plates, films, tubes, and particles. By using particles as a solid, a larger surface area per unit volume can be utilized, so that rapid and efficient processing can be performed. Further, by using magnetized or magnetizable magnetic beads as particles, the separation process and the like can be automated, made efficient, or accelerated. Moreover, when using particle | grains as solid, the diameter of particle | grains is 50 micrometers or less normally, for example, 1.0 micrometer-3.0 micrometers.

A method for immobilizing a nucleic acid such as a target nucleic acid chain on a solid surface is not particularly limited. For example, covalent bond, ionic bond, physical adsorption, biological bond (for example, binding of biotin and avidin or streptavidin, antigen And the like, and the like. The nucleic acid may be immobilized on the solid surface via a spacer sequence, for example a hydrocarbon group containing 1 to 10 carbon atoms.
For example, when the target nucleic acid strand is cDNA, a probe nucleic acid containing a poly T sequence is bound to a solid surface during cDNA synthesis, mRNA is bound to the poly T sequence, and cDNA is synthesized by complementary strand synthesis. The target cDNA can be immobilized on the solid surface via the probe nucleic acid. A probe nucleic acid containing a poly-T sequence can also be bound to a solid surface by the method of immobilizing the nucleic acid on the solid surface as described above.

  Immobilization of a nucleic acid on a solid surface via a covalent bond can be carried out, for example, by introducing a functional group into the nucleic acid and introducing a functional group reactive with the functional group onto the solid surface to cause both to react. For example, a covalent bond can be formed by introducing an amino group into a nucleic acid and introducing an active ester group, epoxy group, aldehyde group, carbodiimide group, isothiocyanate group or isocyanate group onto the solid surface. Further, a mercapto group may be introduced into the nucleic acid, and an active ester group, maleimide group or disulfide group may be introduced onto the solid surface. Examples of the active ester group include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimide group, 5-norbornene-2, and 3-dicarboximide group.

  One method for introducing a functional group onto a solid surface is to treat the solid surface with a silane coupling agent having a desired functional group. Examples of coupling agents include γ-aminopropyltriethoxysilane, N-β- (aminoethyl) -γ-aminopropyltrimethoxysilane, N-β- (aminoethyl) -β-aminopropylmethyldimethoxysilane, Alternatively, γ-glycidoxypropyltrimethoxysilane or the like can be used. As another method for introducing a functional group serving as a binding site to a solid surface, plasma treatment may be mentioned. By such plasma treatment, a functional group such as a hydroxyl group or an amino group can be introduced on the solid surface. The plasma treatment can be performed using an apparatus known to those skilled in the art.

  Examples of a method for immobilizing a nucleic acid on a solid surface by physical adsorption include a method of electrostatically binding a solid surface-treated with a polycation (polylysine, polyallylamine, polyethyleneimine, etc.) using the charge of the nucleic acid. .

  In the present invention, a repetitive sequence is introduced at the 3 ′ end of the target nucleic acid strand prepared as described above. In the present invention, the “repetitive sequence” means a sequence repeatedly containing a repeating unit of a certain length, and in the present invention, a priming site for primer hybridization in an amplification reaction or complementary strand synthesis described later. I will provide a. Accordingly, the repeating unit includes at least one priming site sequence (homologous sequence or complementary sequence), and may optionally include a preliminary priming site sequence, a restriction enzyme cleavage recognition sequence, and the like. The repeating unit can be about 30 to 300 bases long, for example about 50 to 100 bases long, preferably about 70 bases long. The number of repeating units contained in this repeating sequence is about 50 to 10,000, preferably more than 100, more preferably more than 1000. The repetitive sequence may be shorter or longer than the length of the target sequence of the target nucleic acid strand or its complementary sequence.

Further, when a plurality of types of nucleic acid chains are targeted as the target nucleic acid chain, a common repetitive sequence may be introduced or a different repetitive sequence may be introduced into the plurality of types of nucleic acid strands. . It is preferable to introduce a common repetitive sequence into a plurality of types of target nucleic acid strands in that subsequent complementary strand synthesis can be easily performed simultaneously in parallel.
One method for introducing a repetitive sequence is a method of introducing a nucleic acid strand having a repetitive sequence into a target nucleic acid strand by ligation. Ligation can be performed by a method known in the art, for example, a method using a restriction enzyme or ligase. In another method, a nucleic acid chain having a repetitive sequence is used as a template, and the 3 ′ end portion of the target nucleic acid strand is used as a primer for repetitive sequence introduction, or a sequence portion added to the 3 ′ end of the target nucleic acid strand is used as a repetitive sequence introduction primer. In which a nucleic acid chain having a repetitive sequence is introduced into a target nucleic acid chain by complementary strand synthesis used as

  In the method of the present invention, complementary strand synthesis can be performed according to a method known in the art, for example, by using an appropriate polymerase in the presence of a base (such as dNTP) serving as a substrate. In addition, the oligonucleotides functioning as primers and probes used for complementary strand synthesis and rolling circle amplification (RCA) reaction in the method of the present invention are based on the target sequence region to be amplified, based on the sequence to be hybridized, In consideration of the length of the base sequence, the melting temperature (Tm), etc., it can be designed easily using, for example, a known program. For example, the length of the oligonucleotide having a function as a primer or a probe is preferably 10 bases or more, more preferably 15 to 50 bases, and further preferably 20 to 30 bases.

  The nucleic acid chain having a repetitive sequence used in the above method can be prepared by a method known in the art. For example, a nucleic acid strand having a repetitive sequence can be generated by complementary strand synthesis using a ring-shaped nucleic acid as a template. Specifically, a nucleic acid chain having a repetitive sequence can be generated by performing a rolling circle amplification (RCA) reaction using a ring-shaped nucleic acid (for example, ring DNA) composed of a sequence of repetitive units as a template. In the present invention, the “ring-shaped nucleic acid” means a so-called circular nucleic acid, and means not linear. The ring-shaped nucleic acid contains a sequence complementary to the above-mentioned repeating unit, and has a length of about 30 to 300 bases, for example, about 50 to 100 bases, preferably about 70 bases.

When synthesizing a complementary strand using a ring-shaped nucleic acid as a template for the introduction of a repetitive sequence, the number of repetitive units of the repetitive sequence to be introduced constitutes the ring-shaped nucleic acid regardless of the sequence or length of the target nucleic acid. Determined by sequence length and complementary strand synthesis time. Since the time required for synthesizing one base complementary strand is about 2 to 3 msec, when complementary strand synthesis is performed for 15 minutes, a base length of about 2 to 3 × 10 ⁵ is extended as a repetitive sequence. When the size of the ring nucleic acid is about 70 bases, amplification (repetitive sequence) of about 3 to 4 × 10 ³ times is obtained. If this is repeated twice, amplification of about 10 ⁷ times can be obtained in a short time.

  As another method for introducing a repetitive sequence, for example, when the sequence at the end of the target nucleic acid strand is known, a sequence portion contained in the target nucleic acid strand is used as a primer for repetitive sequence introduction, and a ring-shaped nucleic acid is used. Repeated sequences can be introduced by complementary strand synthesis using as a template. The ring-shaped nucleic acid used here is also composed of a sequence of repeating units as described above.

  Alternatively, when the sequence at the end of the target nucleic acid chain is not known, as another method for introducing a repetitive sequence, a sequence portion added to the target nucleic acid chain is used as a primer for repetitive sequence introduction, Repeated sequences can be introduced by complementary strand synthesis using a nucleic acid as a template. In addition, in order to introduce a repetitive sequence, an oligonucleotide that can be used as a repetitive sequence introduction primer using a sequence portion added to a target nucleic acid strand and hybridize to the sequence portion and form a ring-shaped nucleic acid by ligation It is also possible to perform complementary strand synthesis using a ring-shaped nucleic acid formed using as a template.

  The repetitive sequence introduction primer used in the above method utilizes, for example, a sequence portion at least a part of which is added to the 3 ′ end of the target nucleic acid strand. In addition, a sequence portion such as a poly A sequence or a poly C sequence can be added to the 3 ′ end of the target nucleic acid strand by terminal transferase.

  Furthermore, as a method for introducing a repetitive sequence, a complementary sequence of the sequence portion obtained by complementary strand synthesis is repeated using an oligonucleotide that hybridizes to at least a part of the sequence portion added to the 3 ′ end of the target nucleic acid strand as a template. A method for synthesizing a complementary strand using a ring-shaped nucleic acid as a template can be used as a primer for sequence introduction. Here, the sequence portion can be added to the 3 ′ end of the target nucleic acid strand by terminal transferase. Specifically, this method hybridizes to a sequence portion on the 3 ′ end side of the target nucleic acid strand adjacent to the sequence portion added to the 3 ′ end of the target nucleic acid strand and all or part of the added sequence portion. And complementary strand synthesis using an oligonucleotide containing a known sequence as a template, removing part of the sequence portion added to the 3 ′ end of the target nucleic acid strand as necessary, and subsequently hybridizing the oligonucleotide Using the known sequence added to the 3 'end of the target nucleic acid strand as a template for complementary strand synthesis as a primer for repeated sequence introduction, repeat strands are introduced by carrying out complementary strand synthesis using a ring nucleic acid as a template. To do.

  In the above method, the removal of a part of the sequence portion added to the 3 ′ end of the target nucleic acid strand is performed by removing the base of the portion where the oligonucleotide in the sequence portion is not hybridized from the 3 ′ end of the single-stranded nucleic acid. It can be carried out by removing it using an enzyme such as exonuclease (ExoI) that degrades.

  In addition, the sequence portion used as a primer for introducing a repeated sequence added to the 3 ′ end of the target nucleic acid chain during the introduction of the above-described repeated sequence is an oligonucleotide ligation following restriction enzyme cleavage or an oligonucleotide as a template. It can be produced by complementary strand synthesis.

  Alternatively, a known sequence part contained in the target nucleic acid chain can be used as a primer for repetitive sequence introduction. For example, a known sequence portion of the target nucleic acid strand can be cleaved, and the portion containing the 3 ′ end of the target nucleic acid strand obtained can be used repeatedly as a primer for sequence introduction. This cleavage can be performed using, for example, an enzyme such as Cleavase.

  As another method, a complementary strand synthesis is performed in which an oligonucleotide having an anchor sequence at the 5 ′ end is hybridized to a target nucleic acid strand, and then the oligonucleotide is extended. The double-stranded nucleic acid is generated, and the single-stranded portion of the target nucleic acid strand is decomposed and removed using an enzyme that degrades the single-stranded nucleic acid from the 3 ′ end, and then the target nucleic acid strand using the anchor sequence of the oligonucleotide as a template. It is also possible to generate a primer for repetitive sequence introduction by adding a sequence portion to the target nucleic acid strand by synthesizing a complementary strand extending the 3 ′ terminal side.

  Alternatively, a double strand is obtained by hybridizing to a known sequence portion of the target nucleic acid strand and hybridizing an oligonucleotide having an anchor sequence on the 5 ′ end side to the target nucleic acid strand and extending the oligonucleotide to perform complementary strand synthesis. A nucleic acid is generated, a single-stranded portion of the target nucleic acid strand is removed, and then a known sequence portion is added to the 3 ′ end of the target nucleic acid strand by complementary strand synthesis using the anchor sequence of the oligonucleotide as a template. A primer for repetitive sequence introduction can be generated by removing.

  Further, the repeating sequence may be further introduced on the 5 ′ end side of the target nucleic acid strand. For example, a known sequence portion is added to the 3 ′ end and 5 ′ end of the target nucleic acid strand, and the known sequence portion at the 3 ′ end of both the target nucleic acid strand and its complementary nucleic acid strand is repeatedly used as a primer for sequence introduction. By carrying out complementary strand synthesis using a ring-shaped DNA that can hybridize to the sequence portion as a template, it is possible to repeatedly introduce sequences at the 3 ′ end and 5 ′ end.

  Subsequently, using an oligonucleotide that hybridizes to the repetitive sequence introduced into the target nucleic acid strand, a plurality of oligonucleotides are hybridized to the repetitive sequence, and complementary strand synthesis is simultaneously performed.

  As a polymerase to be used for complementary strand synthesis, a type (strand displacement type) polymerase that synthesizes a complementary strand by peeling off the double-stranded portion of the template nucleic acid is used. The polymerase that can be used in the present invention is not particularly limited as long as it has such a strand displacement activity. For example, Bst DNA polymerase (large fragment), Bca (exo-) DNA polymerase, E. coli DNA polymerase I Klenow fragment, Vent (Exo-) DNA polymerase (Vent DNA polymerase minus exonuclease activity), DeepVent (Exo-) DNA polymerase (DeepVent DNA polymerase minus exonuclease activity) and KOD DNA polymerase Etc. Bst DNA polymerase (large fragment) is preferably used. When using Bst DNA polymerase, it is desirable to carry out the reaction at around 60 to 65 ° C., which is the optimum temperature for the reaction.

  When such a strand displacement type polymerase is used, a repetitive sequence is introduced into the end of the target nucleic acid strand and used as a template. A plurality of oligonucleotides hybridize to the repetitive sequence portion as primers, and these are almost Simultaneously, complementary strand synthesis is performed. For example, about 100 to about 100000, for example, about 1000 to 50000 oligonucleotides can be hybridized with each primer as a primer. When a nucleic acid strand generated from one primer extends to a nucleic acid strand generated from another primer hybridized downstream thereof, complementary strand synthesis continues while stripping it. As a result, the nucleic acid chains synthesized starting from the primer hybridized at a position close to the target nucleic acid chain are successively peeled off and released as a single strand. Finally, the primer that hybridizes to the 3 'end of the target nucleic acid strand that is the template finishes complementary strand synthesis to form double-stranded DNA, but hybridizes to a shorter position (position far from the 3' end). All of the nucleic acid strands synthesized by the primers are peeled off and released as single strands.

  A specific complementary strand synthesis step can be performed, for example, as follows. In one method, complementary strand synthesis is performed using a first oligonucleotide that hybridizes to a repetitive sequence introduced into a target nucleic acid strand as a primer, and the resulting complementary nucleic acid strand is used as a template to hybridize to its 3 ′ end. A complementary strand synthesis is performed using the second oligonucleotide as a primer, and a step of amplifying a copy of the target nucleic acid strand is performed. In another method, complementary strand synthesis is performed using, as a primer, an oligonucleotide that hybridizes to a repetitive sequence introduced into the target nucleic acid strand, and the resulting double-stranded nucleic acid is used as a single-stranded nucleic acid. As a primer, the step of performing complementary strand synthesis using the generated single-stranded nucleic acid as a template is repeated.

  The oligonucleotide used here may be immobilized on a solid surface. For example, the first oligonucleotide that hybridizes to the repetitive sequence introduced into the target nucleic acid strand may be immobilized on the solid surface, or the second oligonucleotide that hybridizes to the complementary nucleic acid strand generated by the first complementary strand synthesis. The oligonucleotides may be immobilized on a solid surface, or at least two oligonucleotides (eg, first and second oligonucleotides) may be immobilized on the same or different solid surfaces. For example, when the second oligonucleotide is immobilized on the solid surface, the complementary nucleic acid strand generated using the first oligonucleotide that hybridizes to the repetitive sequence introduced into the target nucleic acid strand is immobilized on the solid surface. Capture is performed using the second oligonucleotide, and complementary strand synthesis is performed using the second oligonucleotide as a primer. As the solid used here, any solid can be used as long as it is generally used for the operation of nucleic acids as described above, and the solid and the oligonucleotide are bound as described above. Can do.

  The method of the present invention also includes, for example, a step of synthesizing a complementary strand using a target nucleic acid strand as a template using a capture probe having a sequence complementary to the target nucleic acid strand and a first anchor sequence at the 5 ′ end, and the generated complementation. Step of making nucleic acid strand as single strand, synthesis of complementary strand using first oligonucleotide that hybridizes to single strand complementary nucleic acid strand, and the first oligonucleotide on the 3 ′ end side where the first oligonucleotide is extended A step of introducing a known sequence portion based on an anchor sequence, a complementary strand synthesis is carried out by hybridizing to a ring-shaped nucleic acid strand using the known sequence portion as a primer, and a complement having the first repetitive sequence introduced at the 3 ′ end side A step of generating a nucleic acid strand, and hybridizing a plurality of second oligonucleotides to the first repetitive sequence to synthesize complementary strands in parallel It can be carried out by step. In this method, by including a second anchor sequence on the 5 ′ end side of the first oligonucleotide, the complementary strand synthesized by using the second oligonucleotide that hybridizes to the first repetitive sequence as a primer is synthesized. A known sequence portion based on the second anchor sequence is introduced at the 3 ′ end side, and the known sequence portion is used as a primer to synthesize a complementary strand using a ring-shaped nucleic acid as a template. The step of generating a complementary nucleic acid strand into which the repetitive sequence is introduced can be further performed, and the complementary strand synthesis can also be performed in parallel from the second repetitive sequence.

  In the above-mentioned complementary strand synthesis, the length of the introduced repetitive sequence does not vary depending on the type or sequence of the target nucleic acid strand, and is almost constant, so that uniform amplification is possible regardless of the type or sequence of the target nucleic acid strand. Become. Further, amplification can be performed at a high amplification rate in a short time. This amplification process can further increase the amplification factor by using linear amplification with excellent amplification efficiency and thermal cycle in combination. For example, at the time of complementary strand synthesis, the target sequence or its complementary sequence is included, the 3 ′ end has a repeat sequence, and the 5 ′ end has a loop sequence (ie, a single-stranded sequence portion that does not form a double strand). By using the oligonucleotide that hybridizes to the loop sequence of the amplified target nucleic acid strand by further performing a step of generating a target nucleic acid strand having The synthesis can be repeated. Further, for example, by introducing a cleaved portion (nick) into a double-stranded nucleic acid generated by complementary strand synthesis, the complementary strand synthesis can be repeated while peeling the double-stranded nucleic acid from the cleaved portion (nick). The cleaving part (nick) recognizes a recognition sequence and uses an enzyme that cleaves one of the two strands (for example, N.BstNBI). The cleaved portion can be formed by designing an oligonucleotide to be used as a primer so as to be introduced into the DNA, introducing the recognition sequence into a double-stranded nucleic acid by complementary strand synthesis, and reacting the enzyme.

  In addition, a repetitive sequence is introduced into the target nucleic acid strand amplified by the above reaction or its complementary strand, and this is a complementary strand so that a restriction enzyme cleavage recognition sequence is introduced before or after the repetitive sequence. By performing the synthesis, it is possible to cleave and remove the repetitive sequence from the complementary nucleic acid strand produced using a restriction enzyme. For example, it is preferable to put such a restriction enzyme cleavage recognition sequence in the repeating unit of the repeating sequence.

  By the above method, nucleic acid amplification can be performed at a high amplification rate in a very short time. This method is effectively used for gene expression analysis (quantitative analysis) that requires uniform amplification and diagnostic tests for diseases such as infectious diseases that require highly efficient nucleic acid amplification.

  As an example of such an application, the present invention relates to a method for detecting a nucleic acid in a sample by amplifying a nucleic acid in a sample using the above amplification method and detecting the amplification product.

  The sample is not particularly limited as long as it is a biological sample containing a nucleic acid to be detected, and any sample such as a cell sample, a tissue sample, or a liquid sample can be used. The organism from which the sample is derived is not particularly limited, and includes vertebrates (eg, mammals, birds, reptiles, fishes, amphibians), invertebrates (eg, insects, nematodes, crustaceans), protists, Samples derived from any living body such as plants, fungi, bacteria, and viruses can be used. Nucleic acid preparation from a sample can be performed as described above.

  Subsequently, the nucleic acid derived from the sample is amplified according to the amplification reaction described above. Here, all the nucleic acids in the sample may be amplified, or only the nucleic acid to be detected may be amplified.

  In order to detect whether or not the nucleic acid to be detected is present, a known means capable of specifically recognizing the obtained amplification product can be used. For example, by labeling an oligonucleotide to be used as a primer during an amplification reaction, hybridizing the obtained amplification product with a probe specific to the nucleic acid to be detected, and detecting the hybridization using the label, Nucleic acids can be detected. Alternatively, the target nucleic acid can be detected by hybridizing the obtained amplification product with a labeled probe specific to the nucleic acid to be detected and detecting the hybridization using the label. Alternatively, a probe that fluoresces when a target sequence such as a molecular beacon is present is known, and the target nucleic acid is detected by detecting an amplification product that specifically hybridizes with such a probe based on the fluorescence. Can be detected.

  Such a hybridization reaction is known in the art, and is a reaction for specifically binding a nucleic acid under stringent conditions. The conditions are also known. When performing hybridization, the nucleic acid amplification product derived from the sample may be immobilized on an appropriate solid surface (slide glass, membrane, microtiter plate, etc.), or the probe may be immobilized on an appropriate solid surface. . When either one is fixed, non-hybridized nucleic acid or the like can be easily removed by washing.

  Alternatively, when only the nucleic acid to be detected contained in the sample is amplified in the amplification method, the target nucleic acid can be detected depending on whether or not an amplification product is obtained. For example, an oligonucleotide used as a primer during the amplification reaction Or by labeling the substrate (base) used for synthesizing the complementary strand, the label is incorporated into the obtained amplification product, and the presence or absence of the amplification product can be detected based on the label.

The label may be any label, and radioisotopes, fluorescent substances, luminescent substances, enzymes, biotin labels, and the like can be used. As the radioisotope, ³² P, ¹²⁵ I, ³⁵ S and the like can be used. As the fluorescent substance, for example, fluorescein isothiocyanate (FITC), sulforhodamine (SR), tetramethylrhodamine (TRITC) and the like can be used. As the luminescent substance, luciferin or the like can be used. As the enzyme, β-galactosidase or the like can be used. There are no particular restrictions on the type of the labeled body, the method for introducing the labeled body, and the like, and various conventionally known means can be used. For example, as a method for introducing a label, a random prime method using a radioisotope can be mentioned.

  The detection based on the label may be any method known in the art for detecting the above-described labeling body. For example, when a radioisotope is used as the label, the radioactivity can be measured using, for example, a liquid scintillation counter or a γ-counter. In addition, when fluorescence is used as the label, the fluorescence can be detected using a fluorescence microscope, a fluorescence plate reader, or the like. When the label is detected, the nucleic acid to be detected is present in the sample.

  In addition, quantitative detection is possible by using the concentration of the label as an index. Examples of the detection method using a labeled probe include Southern hybridization method and Northern hybridization method.

  Further, in the amplification method described above, when the amplification factor is high, the presence or absence of the nucleic acid to be detected can also be known by measuring turbidity.

  Furthermore, the amplification product obtained can be subdivided, and the expression of the nucleic acid to be detected can be quantitatively analyzed by real-time PCR (Taqman) or the like.

  The nucleic acid amplification method of the present invention is excellent as a method for uniformly amplifying nucleic acids, and in particular, converting a small amount of mRNA obtained from one cell to cDNA and amplifying them uniformly without bias. Further, by repeating the amplification operation, Since many nucleic acid copies can be obtained in a short time, it is also effective for rapid DNA testing required for infectious disease testing.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples do not limit the present invention.

[Example 1] Method of adding a known sequence to the end of a target cDNA for the introduction of a repetitive sequence This example is an example of uniformly amplifying cDNA contained in a cDNA library derived from one cell. The outline of the method is shown in FIG. According to the literature (Nature Method 6, 503-506 (2009)), a cDNA library is prepared using magnetic beads to which a probe comprising an anchor sequence and a poly-T sequence is immobilized. That is, as shown in (1) of FIG. 1-1, mRNA was captured using Dynabeads having a diameter of 1 μm with an mRNA capture probe consisting of anchor sequence 1 and poly T sequence immobilized on the surface, followed by cDNA synthesis. And a cDNA library in which the 5 ′ end of the cDNA is fixed to the beads is obtained. Here, as the poly-T sequence, a sequence in which 3 G bases were inserted every 5 bases was used. This was introduced later for the purpose of increasing Tm in order to facilitate the complementary strand synthesis of poly T sequence when cDNA was used as a template for complementary strand synthesis. The cDNA synthesis of this poly-T sequence part is sufficiently low in temperature and proceeds without problems, but in the complementary strand synthesis using this as a template in the subsequent step, the temperature is slightly higher, and the double-stranded DNA in the lower Tm part is It is possible to avoid interruption of complementary strand synthesis due to separation.

  After cDNA synthesis, washing is performed to remove unnecessary reagents, and then a known sequence is added to the 3 ′ end. This can be accomplished by adding a sequence such as poly A or poly C using terminal transferase, or by hybridizing a double-stranded oligomer with protruding 3 bases on the 3 'side to the end to synthesize the complementary strand of the oligomer. Subsequently, there is a method of adding a known sequence to the 3 ′ end by ligation. There is also a method of adding a known sequence by binding an oligomer having a known sequence by ligation to the cut end cleaved with a restriction enzyme. Here, a poly C sequence is added to the end of the target DNA, followed by a 3 base random sequence at the 3 ′ end, followed by a known sequence at the end using a random primer having a 4 base G sequence and an anchor sequence 2. An example using the method of adding is described. Here, a primer consisting of a mixture of random sequences consisting of 3 bases (total of 64 types, but the 3rd base can be other than G base, so 48 types) was used. It is also possible to change the GGGG sequence to GGG by lengthening the random sequence portion. This is not the case because other methods can be used as well.

  In this example, as shown in (2) of FIG. 1-1, an example in which 5 to 10 C bases (CCCCCCC in FIG. 1-1) were added to the terminal using a terminal transferase was shown. Subsequently, as shown in (3) of FIG. 1-1, the primer hybridizes with a random sequence in the cDNA and the CCCC portion that follows, and performs complementary strand synthesis. Next, as shown in Fig. 1-1 (4), an exonuclease (ExoI enzyme: an enzyme that cleaves a single strand from the 3 'end) is added to the reaction solution, and one strand on the 3' end side of the cDNA is added. The strand part is cut and removed. When the single-stranded portion is removed, the 3 ′ end of the target cDNA becomes hybridized with the DNA strand having the anchor sequence 2, and complementary strand synthesis is possible using this as a template. Thus, a sequence complementary to the anchor sequence 2 can be added to the end of the target cDNA. This part functions as a primer P1-1 in complementary strand synthesis using ring DNA as a template in the subsequent reaction. In addition, when generating cDNA from mRNA, using a reverse transcriptase derived from Moloney Murine Leukemia Virus having terminal transferase activity (for example, SuperScript II) is advantageous because it can introduce CCC of about 3 bases at the end. . In this case, a known sequence can be added by introducing a double-stranded DNA having a complementary strand hybridized to the anchor sequence portion into the end of the target cDNA by a ligation reaction.

  Here, if the target cDNA is obtained in a form immobilized on magnetic beads, it is convenient for purification. After introducing the known sequence primer P1-1 at the end, the extraneous coexistence is removed leaving the cDNA captured on the magnetic beads ((1) in FIG. 1-1). Next, as shown in FIG. 1-1 (7), the target cDNA is a ring DNA consisting of about 70 bases of single-stranded DNA, a nucleic acid substrate, a strand displacement type DNA polymerase (for example, Bst DNA polymerase, φ29 DNA polymerase). ) Etc. are added to synthesize complementary strands. Here, φ29 DNA polymerase operating at approximately 30 ° C. is used. A smaller ring DNA is advantageous for amplification, and a ring DNA having a sequence much shorter than the length of the target DNA is usually used. As shown in Fig. 1-2, the ring DNA is composed of a region (approximately 20 bases) where primer P1-1 hybridizes, a restriction enzyme cleavage recognition sequence, and a DNA strand generated by second primer P1-2. It includes a region having a homologous sequence with primer P1-2 for producing priming site 1-2 for soybean, and a preliminary priming site 1-4. The arrow in FIG. 1-1 (8) indicates the 3 ′ side for reference. Primer P1-2 hybridizes in the opposite direction and extends in the direction opposite to the arrow. The restriction enzyme cleavage recognition sequence should be a sequence that does not exist in mRNA as much as possible, and a recognition sequence by a rare cutter restriction enzyme having a long recognition sequence is preferably used. Such rare cutter restriction enzymes include 6-base recognition enzyme Bam HI (recognition sequence 5'-G | GATCC --- 3 ') or 8-base recognition enzyme Sda I (recognition sequence 5' --- CCTGCA | GG-- -3 '), but not limited to this.

  The number of repetitive sequences to be introduced depends on the purpose, but when amplifying all target DNAs to increase the number of copies and increasing the number of copies, and then performing quantitative PCR by dividing the solution, it is about several hundreds. What is necessary is just to be able to introduce a repeating unit. On the other hand, when it is desirable to achieve a large amplification factor, the unit of the repetitive sequence to be introduced is preferably several thousand or more. Furthermore, the number of introduced units of the repetitive sequence can be increased by introducing a repetitive sequence at both ends of 3 ′ and 5 ′ or repeating the amplification operation.

  Here, complementary strand synthesis was carried out for 5 minutes using ring DNA as a template for introducing repetitive sequences ((7) in FIG. 1-1). Complementary strand synthesis using ring DNA as a template proceeds to synthesize complementary strands of the same ring sequence for all target cDNAs regardless of the cDNA sequence. The length of the repetitive sequence introduced into the end of the target cDNA in a certain time is almost the same regardless of the type of cDNA. Since the speed of DNA complementary strand synthesis is about 50 to 300 bases per second, several hundred to 1,000 repetitive sequence units can be introduced in 5 minutes. After the introduction of the repetitive sequence by complementary strand synthesis, the ring DNA is removed and the repetitive sequence portion consisting of the complementary strand of the ring DNA (primer P1-1 sequence, restriction enzyme cleavage recognition sequence, priming site 1-2 sequence and spare sequence) Primer P1-2 that hybridizes to priming site 1-2 (consisting of the sequence of priming site 1-4) is added to synthesize complementary strands ((8) in FIG. 1-1). About 1000 primers P1-2 hybridize to a repetitive sequence introduced into one target cDNA and synthesize complementary strands in parallel. When complementary strand synthesis is performed for about 10 minutes, complementary strand synthesis from primer P1-2 that has hybridized to the 3 'end of the repetitive sequence is completed, and hybridizes to the target cDNA (5' end). The complementary strand generated from the primer P1-2 was stripped and released into the solution in a single-stranded state ((9) in FIG. 1-1). Here, since the capture probe having the anchor sequence 1 and the poly-T sequence was used when preparing the target cDNA ((1) in FIG. 1-1), the generated complementary strand also contains the same sequence. This can be captured with an extra capture probe immobilized on the beads. In addition, after capture, the capture probe is extended using the complementary strand as a template, so that it is held in a double-stranded state on the surface of the bead ((10) in FIG. 1-1). By the above operation, the cDNA library amplified about 1000 times is held on the bead surface.

In this state, restriction sequences may be used to cleave and remove excess sequences (for example, introduced repetitive sequences), and the complementary strand hybridized to the target cDNA may be used for various measurements. To increase it, subject it to thermal cycling and repeat complementary strand synthesis. Since this process can be amplified 1000 times in about 5 minutes, 1000 × 1000/2 = 0.5 × 10 ⁶ copy amplification can be performed in two complementary strand synthesis processes. Furthermore, if complementary strand synthesis is performed three times by thermal cycling, about 10 ⁸ copy amplifications can be realized. Even when such amplification is performed, the required time is 30 minutes or less including the time of thermal divergence and annealing, and it is possible to efficiently amplify a plurality of DNAs simultaneously and uniformly.

  In this example, a ring circle amplification (RCA) reaction was performed using a ring DNA having a known sequence prepared in advance as a template. After removing the ring DNA, primer P1-2 was added and complementary strand synthesis was performed again. From this, primer P1-2 may be added to the reaction system, and a repetitive sequence may be introduced at the end of the target cDNA, and at the same time, complementary strand synthesis from primer P1-2 may proceed simultaneously. In this case, the amount of the ring DNA may be about 0.1 to 1 pmole, but the amount of the second primer P1-2 may be several to 10 pmole. In addition, ring DNA was prepared and used in advance, but the ring DNA was ligated by ligation after hybridization with a probe that hybridizes to the primer P1-1 part at the end of the target cDNA at the 3 'end and 5' end. It may be formed. Here, an oligomer subsequent to the sequence portion added by terminal transferase is introduced as a primer sequence, but the sequence portion introduced by terminal transferase may be used as primer P1-2 (a primer for introducing a repeated sequence).

  Since this method can amplify samples containing many types of DNA simultaneously and uniformly, it is effective for quantitative analysis combined with various measurement methods.

[Example 2] Method using Cleavase In Example 1, a method for uniformly amplifying all DNA in a DNA pool (including a cDNA library) consisting of various DNA fragments was disclosed. A method for uniformly amplifying DNA is disclosed. Of course, it can be applied to single DNA amplification.

  An outline of this example is shown in FIG. The cDNA library is used again as a sample. The cDNA library contains the one that was able to synthesize complementary strands to the end of the mRNA during the preparation process, and the one that the complementary strand synthesis stopped halfway, and should contain various lengths of cDNA even from the same mRNA. ((1) in Fig. 2-1). For this reason, the terminal sequence cannot be treated as having a known sequence. However, it is not clear how far the cDNA synthesis of individual cDNAs has progressed, but the entire sequence is already known. Here, an example in which these genes are uniformly amplified by focusing on 10 kinds of genes will be disclosed. Since cDNA synthesis usually synthesizes complementary strands up to 500 bases without hindrance, select a specific sequence within 500 bases from the poly T site of the cDNA library and probe P2- Make 1 Probe P2-1 comprises a sequence portion that hybridizes to the target cDNA and an anchor sequence portion. The anchor sequence part is designed to self-hybridize to form a loop structure and to hybridize to the target cDNA in a triple chain only at one base site ((2) in FIG. 2-1).

  To a solution containing single-stranded cDNA, a probe P2-1 for a plurality of target genes (probe P2-1 that differs by the number of target genes is necessary) is added and hybridized to the target cDNA. Next, the cleavage enzyme Cleavase is added to cleave the site where the probe P2-1 forms a triple chain with the target cDNA ((3) in FIG. 2-1). This reaction is the same as the enzyme cleavage reaction used in the invader assay used in genetic testing. Of the cleaved cDNA fragments, the fragments on the side fixed to the beads are recovered and used. The sequence following the cleavage site is known with reference to the mRNA database, and the known sequence is introduced using an oligomer (probe P2-2) with an anchor sequence (known sequence) that hybridizes therewith (FIG. 2-1). (4)). That is, a known sequence can be introduced only into the target cDNA species by this process. Of course, a part of the target cDNA sequence may be used as a primer to repeat the amplification reaction using the ring DNA as a template, and in this case, only the number of corresponding genes in the ring DNA sequence may be introduced. It is necessary to create priming sites with different sequences. Here, a sequence complementary to the anchor sequence portion of the probe P2-2 is first synthesized by complementary strand synthesis, and this portion is repeatedly used as a primer for sequence introduction. As shown in (2) of FIG. 2-1, complementary strand synthesis was performed using this sequence portion as a primer and ring DNA as a template, and a repetitive sequence was introduced into the 3 ′ end of the cDNA in the same manner as in Example 1. As described in Example 1, the target cDNA can be uniformly amplified using the probe P2-3 that hybridizes to this repetitive portion. Amplified products can be recovered by beads immobilized cDNA as in Example 1. When it is desirable to remove the amplification product by fixing it to a bead different from the beads to which the cDNA library is immobilized, or in a solution state, a capture probe (mRNA consisting of anchor sequence and poly T sequence immobilized on the bead) If the probe P2-4, which has the same sequence as that used to capture the DNA) and is not immobilized, is added, the released synthetic complementary strand hybridizes to the probe P2-4 and is not immobilized. This produces a chain that can be recovered. At this time, if a probe with biotin is used as the probe P2-4, the generated DNA strand can be easily recovered.

  In this example, following cleavage with Cleavase, probe P2-2 having a known sequence was hybridized to the end of the target cDNA and a known sequence was added by complementary strand synthesis. However, the sequence of the cleavage end site is known. This part may be designed to hybridize to the ring DNA. In this case, the same number of ring DNAs as the target type is required, but it is also possible to introduce a priming site corresponding to a plurality of types of target cDNAs into the ring DNA and use only one type of ring DNA. In this case, in addition to the above priming sites, the ring DNA contains a restriction enzyme cleavage recognition sequence and a priming site group for complementary strand synthesis (primer P2-3 homologous sequence region and spare priming site 2-5 region). ) Is included (FIG. 2-2).

  This process may be repeated when there are many types of genes of interest, but the uniformity of the amplification factor is higher when performed at once.

  In this way, a large number of priming sites for synthesizing complementary strands using the target DNA as a template are introduced by the RCA reaction, and then complementary strand synthesis is simultaneously performed in parallel from each priming site, enabling uniform and efficient amplification. realizable. If the same sequence as the target DNA is required, it can be handled by synthesizing the complementary strand again using the synthesized complementary strand as a template.

  In the above example, a loop-shaped probe that hybridizes to a known sequence portion of the target DNA and Cleavase are used to cleave the target DNA at the known sequence portion to make the end portion a known sequence, and then an anchored probe is hybridized here. Complementary strand synthesis was carried out using soybean, and a common known sequence was introduced at the ends. As another example, a probe with an anchor is first hybridized to the target DNA, a complementary strand synthesis is performed to form a stable duplex, and a single strand from the 3 ′ end such as an exonuclease (ExoI, etc.) There is an example using an enzyme that cleaves DNA. The target DNA has a fixed 5 ′ end. When the 3 ′ end is in a single-stranded state, the single-stranded portion is decomposed and removed. That is, after hybridizing the anchored probe P2-2 to the target cDNA and stabilizing the double-stranded portion by complementary strand synthesis, two target cDNA strands including the remaining 3 ′ end are present from the 3 ′ end side. The sequence of the primer P2-1 can also be introduced by decomposing to the strand part and synthesizing the complementary strand.

[Example 3]
In Examples 1 and 2, the priming region was introduced by repeatedly introducing a sequence into the 3 ′ end of the target cDNA to amplify the entire region up to the 5 ′ end of the target DNA. A method for amplifying a region is disclosed. This is advantageous when the target DNA is very long. In addition, a short DNA containing the target sequence to be amplified is prepared from the target DNA. A known sequence is introduced into both ends of this DNA, and this is used as a primer to introduce repeated sequences at both ends to create a large number of priming sites. Also disclosed is a technique for significantly increasing the amplification factor by synthesizing complementary strands from these priming sites.

  The outline is shown in Fig. 3-1. First, hybridize outside the primer P3-1 with anchor sequence 3-1a and primer P3-2 with anchor sequence 3-2a that hybridizes to the target DNA and its complementary DNA across the amplification region of the target DNA. Prepare primers P3-3 and P3-4. The target may be a double-stranded DNA, but here it is a single-stranded cDNA. The amounts of primer P3-1 and primer P3-3 were 1 pmol and 0.2 pmol, respectively. The reason for reducing the amount of primer P3-3 is to allow primer P3-1 to hybridize more rapidly to the target DNA. These primers are prepared only for the type of target DNA, but the anchor sequence portion 3-1a is common.

  As shown in (1) of FIG. 3-1, primer P3-1 and primer P3-3 hybridize to the target DNA to synthesize complementary strands. Primer P3-3 synthesizes the complementary strand while peeling off the DNA strand (DNA3-1) produced by primer P3-1 and releases the complementary strand (DNA3-1) starting from primer P3-1 as a single strand ((2) in FIG. 3-1). As shown in (3) of FIG. 3-1, the complementary strand DNA 3-1 released as a single strand is hybridized with the primer P3-2 and the primer P3-4 to synthesize a complementary strand. The primer P3-4 plays the role of releasing the DNA strand (DNA3-2) generated from the primer P3-2 as a starting point in the same manner as the primer P3-3 ((4) in FIG. 3-1).

  A sequence complementary to the anchor sequence 3-1a (referred to as “primer sequence P3-1ac”) is present at the 3 ′ end of the single-stranded DNA (DNA3-2) starting from the primer P3-2 thus prepared. It has been added. The primer P3-1ac hybridizes with the anchor sequence 3-1a of the primer P3-1. Therefore, as shown in FIG. 3-1 (5), ring DNA3-1 containing the same sequence is added to the reaction solution in advance. In addition to the anchor sequence 3-1a, a sequence of primer P3-5 and a restriction enzyme cleavage recognition sequence used for synthesizing a strand complementary to DNA 3-2 are included in the sequence of ring DNA3-1. The DNA strand (DNA3-2) starting from the released primer P3-2 hybridizes to the ring DNA3-1 at the 3 'end to synthesize a complementary strand, and a repetitive sequence is introduced to the 3' end (Fig. 3-1 (6)). Since the sequence that hybridizes with primer P3-5 appears repeatedly in the repetitive sequence, complementary strand synthesis occurs at the same time when primer P3-5 is added to the reaction system, and it contains the same sequence as DNA3-1 and has a repetitive sequence. Many DNA strands (DNA3-5) are synthesized and released (FIG. 3-1, (7)). The end of this free DNA strand (DNA3-5) is complementary to the anchor sequence 3-2a. Therefore, as shown in (8) of FIG. 3-1, when a ring DNA 3-2 having an anchor sequence 3-2a is added, DNA 3-5 hybridizes to it as a primer (P3-6). Functions and adds a second repetitive sequence to the synthetic DNA strand (DNA3-5) ((9) in FIG. 3-1). In the same way as the ring DNA 3-1, the sequence of the primer P3-5 is put in the ring DNA 3-2 in advance. Then, a priming site to which primer P3-5 hybridizes is also created in DNA3-5, so that complementary strand synthesis further proceeds, and DNA3-5c having a sequence complementary to DNA3-5 is generated (FIG. 3-1). (Ten)). Since this DNA3-5c contains the priming site of primer P3-5, synthetic DNA strands are generated one after another using this as a template.

As described above, DNAs (DNA3-5 and DNA3-5c) containing the DNA3-2 sequence or its complementary sequence and having a repeat sequence at the 3 ′ end are generated one after another from the primer P3-5 (FIG. 3-1). (10) to (12)). This reaction is repeated and DNA is amplified in a very short time. In addition, the amplification factor when a repetitive sequence is introduced at both ends increases approximately n times each time the reaction at both ends proceeds, assuming that the amplification rate is n times on one side (for example, the 3 ′ end). Furthermore, when the temperature of the reaction solution containing the double-stranded DNA group generated is increased to a single strand and then the above reaction is repeated again (thermal cycle reaction), the amplification rate is 10 ¹⁰ times or more in a short period of 10 to 20 minutes. It is also possible to obtain This is an important advantage for applications that require large DNA amplification in a short time, such as infectious disease diagnosis.

  As a modification of the above embodiment, the present invention can also be implemented by the following method (FIG. 3-2).

  The target group is a group of DNAs having a known sequence at the 5 ′ end, such as a cDNA library prepared from mRNA. A cDNA will be described as an example. As shown in Fig. 3-2 (1), a capture probe in which mRNA is immobilized on a bead (having an anchor sequence + poly-T sequence or a sequence in which G base is mixed with part of the poly-T sequence ) To obtain a cDNA having a capture sequence. Of course, a floating probe may be used instead of a fixed probe.

  Primers P3-01 hybridizing to the target cDNA and having the anchor sequence 3-01a are prepared for the target type. This primer P3-01 may be the same as the primer P2-2 in Example 2. As shown in (2) of FIG. 3-2, this is hybridized to the target cDNA and complementary strand synthesis is performed. A sequence complementary to the capture probe is formed at the 3 ′ end of the synthesized DNA strand. After synthesizing the complementary strand, the temperature is raised, the double-stranded DNA is separated into single strands, and the supernatant is collected ((3) in FIG. 3-2). Here, when a floating probe is used instead of a fixed probe at the time of cDNA preparation, a probe corresponding to the probe P3-3 of this example can be added to the reaction system to release the synthesized DNA from the template DNA.

  As shown in (4) of FIG. 3-2, ring DNA 3-01 having the same sequence as the anchor sequence of the capture probe and φ29 DNA polymerase are added to this solution to synthesize complementary strands. Since the 3 ′ end of the complementary strand DNA (DNA3-01) synthesized in the first reaction contains a capture sequence, it hybridizes to the ring DNA3-01. As a result, an RCA reaction occurs, and a complementary strand having many priming site sequences 3-02 is generated ((5) in FIG. 3-2). At this time, when primer P3-02 that hybridizes to priming site 3-02 is added, it hybridizes to the priming site sequence (3-02) that appears repeatedly in DNA3-01 synthesized by RCA, and performs complementary strand synthesis. Since it starts, many DNA copies (DNA3-02) having the same sequence as the target cDNA can be obtained ((6) in FIG. 3-2). This DNA 3-02 has a sequence (3-01ac) complementary to the anchor sequence 3-01a at the 3 ′ end, and if a ring DNA 3-02 hybridizing thereto is prepared, the second DNA 3-02 is present at the end. Thus, the amplification factor can be further increased ((7) to (9) in FIG. 3-2). In the obtained DNA, a repeated sequence portion is introduced. A restriction enzyme cleavage recognition sequence is put in the ring DNA, a double strand is formed in the same manner as in Example 1, and this portion is cleaved and removed with a restriction enzyme. It is possible.

  In addition, when a plurality of priming sites are included in the ring DNA, a plurality of priming sites are formed in one repetitive sequence, and the amplification factor can be further increased accordingly. In particular, in the method of introducing repetitive sequences on both sides, amplification is increased because the number of units of repetitive sequences is amplified to the second or third power.

[Example 4]
In this example, a loop sequence is introduced on the 5 ′ end side of the DNA sequence portion to be amplified and a repeat sequence is introduced on the 3 ′ end side. The outline is shown in Fig. 4-1. Methods for introducing a repeat sequence at the 3 'end include introducing a known sequence at the end and introducing it by complementary strand synthesis using a ring DNA prepared in advance as a template, or using a ring DNA in advance. There is a method in which a DNA strand having a sequence is synthesized and a complementary strand is synthesized using this as a template. Of course, a DNA strand having such a repetitive sequence may be bound to the target DNA by ligation. In this example, a DNA fragment containing the sequence to be amplified by complementary strand synthesis is prepared using a primer having a loop sequence, and a repetitive sequence is introduced using ring DNA as a template using a known sequence at the end. Is disclosed. Further, a loop sequence is linked to the 5 ′ end of the target DNA to be amplified. This is also performed by hybridizing a probe having the loop sequence as an anchor sequence to the target DNA and synthesizing complementary strands. As shown in -1, loop sequences can be easily linked. In addition, in a sample obtained by synthesizing cDNA from mRNA, it is also possible to put a loop sequence at the end of the probe that captures mRNA.

  Here, as shown in FIGS. 4-1 (1) and (2), a strand displacement type complementation using a probe P4-1 having an anchor sequence in the target DNA and a probe P4-3 that hybridizes to the outside thereof. The chain synthesis is performed to start by releasing a DNA chain (DNA4-1) starting from the probe P4-1 having the anchor sequence. The anchor sequence 4-1a includes the same sequence in the ring DNA 4-1 used as a template, which is used in the reaction described later, and is used to introduce a repeated sequence in a rolling circle amplification (RCA) reaction. Primer P4-2 having a loop sequence 4-2 loop is hybridized to this complementary DNA 4-1 to synthesize a complementary strand again ((3) in FIG. 4-1), having the same sequence as the target DNA, and At the 3 'end is a primer sequence 4-1ac (complementary to the anchor sequence 4-1a) that hybridizes to the ring DNA 4-1, and at the 5' end a template DNA 4-2 having a loop sequence 4-2 loop. Make it ((4) in Figure 4-1). This becomes the template DNA for amplification. There may be a plurality of types of target DNA, and the sequence of the portion that hybridizes to the target DNA differs depending on the type, but the same anchor sequence and loop sequence are used. When cDNA is synthesized from mRNA, the subsequent operation is the same if a capture probe with a loop sequence added is used. The Tm of the loop sequence is preferably designed at a temperature that is the same as or slightly lower than the optimum temperature for strand-displacement complementary strand synthesis. When the complementary strand synthesis of this part is performed, a loop sequence is formed with a high probability, and the synthesized complementary strand is designed to perform complementary strand synthesis using itself as a template.

  FIG. 4A shows an outline of DNA amplification according to this example. As shown in (1) of FIG. 4-1, a probe P4-1 having an anchor sequence 4-1a on the target DNA and a probe P4-3 on the outside thereof are hybridized to obtain a strand displacement type DNA polymerase (φ29 or Bst polymerase). Etc.) to synthesize complementary strands. The complementary strand generated with the probe P4-3 as a starting point serves to peel off the DNA complementary strand (DNA4-1) synthesized with the probe P4-1 as a starting point. As a result, single-stranded DNA 4-1 is released as shown in (2) of FIG. Probe P4-2 and probe P4-4 having a sequence hybridizing to DNA4-1 and a loop sequence 4-2loop are hybridized to DNA4-1 to synthesize complementary strands ((3) in FIG. 4-1). . As before, DNA4-2 starting from probe P4-2 is released. DNA4-2 has a primer sequence 4-1ac (sequence complementary to the anchor sequence) that hybridizes to the ring DNA at the 3 ′ end, contains a sequence to be amplified, and has a loop sequence at the 5 ′ end (Template DNA for amplification) ((4) in FIG. 4A). As shown in (5) of FIG. 4-1, this DNA4-2 hybridizes to the ring DNA4-1 to synthesize a complementary strand, and generates a repeated sequence at the 3 ′ end. A plurality of primers P4-5 are hybridized to the priming site of this repetitive portion, and complementary strand synthesis is carried out simultaneously in parallel ((6) in FIG. 4A). The resulting complementary strand forms a single-stranded DNA 4-5 in a form that is stripped from the template DNA by the synthetic strand that comes later, but the 3 'end portion of the complementary strand synthesis is looped and self-hybridized. As a result, the synthesis of the complementary strand further proceeds to produce double-stranded DNA ((7) in FIG. 4-1). The loop part has a single-stranded state, and as shown in (8) of FIG. 4-1, when primer P4-6 that hybridizes to this region is added, it hybridizes to the loop sequence in the middle part. Initiate complementary strand synthesis. New complementary strand synthesis proceeds in the form of peeling off the already completed double-stranded DNA 4-5. As a result, the 3'-end repeat sequence becomes a single strand and becomes double-stranded from the loop sequence to the 5 'end. DNA strands are created. Probe P4-5 is further hybridized to the single-stranded portion containing the repetitive sequence, and complementary strand synthesis is repeated ((9) and (10) in FIG. 4A). Although the longest strand forms a double strand and is not torn off by a DNA strand that is synthesized later, the loop sequence at the end is in an equilibrium state between the double strand state and the loop state. Primer P4-6 hybridizes to the loop state, and complementary strand synthesis proceeds. That is, the product can automatically continue the complementary strand synthesis reaction indefinitely, and a large amplification efficiency can be obtained. Amplification of DNA having such a loop sequence is also performed by a LAMP (Loop-Mediated Isothermal Amplification) method or the like. However, in the method of the present invention, since a repetitive sequence is introduced, nucleic acids are amplified simultaneously in parallel. It can be said that it is a method with a higher amplification factor in that it can be done.

[Example 5]
In this example, an average of 1 or less cDNA is prepared per bead and its copy is amplified on the same bead. The outline is shown in Fig. 5-1. Probe P5-2 that hybridizes to the repetitive sequence used in this example is immobilized on the solid surface. A cDNA library is prepared using a capture probe immobilized on beads as in Example 1 ((1) in FIG. 5A). Dynabeads having a diameter of 1 μm were used as the beads. Although it is said that the copy number of mRNA in one cell is about 10 ^6, a cDNA library is prepared using 10 ⁸ beads far larger than that. In this case, the probability that a plurality of cDNAs can be formed on one bead becomes extremely small.

In the same manner as in Example 1, as shown in FIGS. 5-1 (2) to (5), a primer (primer P5-1) having an anchor sequence 5-1a at the 3 ′ end of the target DNA was hybridized. A sequence 5-1ac (priming site 5-1ac) complementary to this anchor sequence 5-1a is introduced at the 3 ′ end. Next, an RCA reaction is carried out for about 10 minutes using a ring DNA5-1 of about 70 bases containing the anchor sequence 5-1a, the homologous sequence portion of the probe P5-2, and the restriction enzyme cleavage recognition sequence as a template (FIG. 5). 1 (6)). As a result, many priming sites 5-2c (sites where probe P5-2 hybridizes) are introduced as repeat sequences at the 3 ′ end of the cDNA. In this case, the base length generated by complementary strand synthesis is about 2 × 10 ⁵ and the number of priming sites 5-2c is about 3000. The length of the extended target cDNA strand is about 60 μm assuming that the interval of one base is 0.3 nm, but since it is not actually stretched, it is expected to be 10 microns or less. In this way, the bead-fixed cDNA strand is made into a long cDNA containing a repetitive sequence synthesized with a complementary strand (with a repetitive sequence introduced at the end), and then placed in a reaction chamber with a wide bottom area for subsequent reactions. .

10 When ⁸ beads are densely arranged, they fit in a 10 mm square area, but a slightly wider 30 mm square reaction chamber is used. The reaction chamber is about 0.5 mm thick, and the solution is injected from the side. The probe P5-2 is fixed densely on the bottom (one probe density of 30 to 100 nm ² ) ((7) in FIG. 5A). A magnet can be attached to the upper part and the lower part, and magnetic beads can be brought into close contact with the upper part or the lower part as required. When the reaction solution is left in the reaction chamber or precipitated at the bottom using a magnet, the probe P5-2 is inserted into the target cDNA as shown in (7) of FIG. 5-1. Therefore, the beads having cDNA are firmly captured on the bottom surface by a plurality of probes. Beads that are not bound to cDNA are not captured on the bottom surface, and can be lifted by applying a magnet to the top, which is washed away with the solution, leaving only the beads with cDNA in the reaction chamber. The number of remaining beads is about average ^106, there would be one bead to 30μm square average. As shown in (8) of FIG. 5-1, by complementary strand synthesis starting from probe P5-2, first, complementary strand synthesis proceeds simultaneously from 3000 priming sites, and about 3000 Although a strand DNA (DNA5-2) is produced, there are many capture probes fixed to the beads near the 3 ′ end, and these are hybridized to proceed with complementary strand synthesis from the capture probe. As a result, many double-stranded DNAs are generated to bridge between the bottom surface and the beads. In this state, the temperature is raised so that the DNA strand becomes a single strand, and then the beads are captured again by the probe P5-2. At this stage, the cDNA immobilized on the beads is 3000 times the original amount, and the number of priming sites to which the probe P5-2 hybridizes is increased to about 4 × 10 ^{6 by} calculation. The reason why it is not simply squared is that there are DNA templates of repetitive sequence portions of various lengths. Although it is not actually calculated, it can be estimated that about 10 ⁶ priming sites are generated.

Complementary strand synthesis using probe P5-2 in the same manner as above generates about 10 ⁶ complementary strand copies of cDNA (DNA5-2) on the solid surface, followed by complementary strand synthesis on the beads. The same amount of cDNA copy can be made. By repeating this process, an enormous number of copies can be generated from one copy on the same bead or in the vicinity of the plane position where the bead was captured. First, a repetitive sequence is introduced into the 3 ′ end of cDNA by RCA reaction. If this repetitive sequence is too long, cross reaction may occur between different DNAs. In some cases, it is convenient to repeat the amplification process with the length of the repetitive sequence portion introduced first being about 10 μm (for example, the repeat unit of 70 bases is increased about 500 times).

  As described above, the amplification method by introducing a repetitive sequence makes it possible to spatially limit the region involved in amplification by utilizing a probe immobilized on a solid surface, so that it is created based on one DNA molecule. The copied copy can be fixed and taken out on a spatially separated material such as beads.

  Furthermore, depending on the measurement, it may be desirable to efficiently prepare a cDNA library with small beads, and to perform various measurements after amplification by transferring the amplified product to large beads that are relatively easy to operate. In such a case, a localized DNA complementary strand (DNA5-2) prepared on the surface to which the probe P5-2 is immobilized may be used. In the above example, it is generated in a state where 3000 complementary strands are localized from one bead. A complementary sequence to the capture probe is added to the 3 ′ end of the complementary strand. Therefore, the temperature is raised, and the beads are washed away after the double-stranded DNA formed between the beads on which the cDNA is immobilized and the solid surface are separated. Next, a large bead on which the capture probe is immobilized is poured into the reaction chamber, and the capture probe is hybridized to the DNA strand having a sequence complementary to the capture probe at the tip generated by the extension of probe P5-2 and held on the bead. After that, excess beads may be washed away and the same operation as described above may be performed. Thus, this method is an excellent DNA amplification method that can be used in various ways.

  In this example, target DNA was first prepared on beads and used, but free DNA can also be targeted. In this case, instead of the probe immobilized on the bead (capture probe), a probe having the same sequence is added to the reaction solution in a free state, or the probe P5- is immobilized on the solid surface on which the probe P5-2 is immobilized. For example, the capture probe P5-3 may be fixed at a fixed ratio with respect to 2 ((a) in FIG. 5-2). Furthermore, in another embodiment, as shown in FIG. 5-2 (b), the reaction chamber can be formed of an assembly of fine reaction cells (pore array). Each reaction cell should contain no more than one target DNA on average. An example is shown in which the probe P5-2 is fixed in the reaction cell, and the probe P5-3 corresponding to the capture probe is present in the solution in a suspended state. A large number of probes P5-2 are hybridized to the repetitive sequence portion of the target DNA, and complementary strand synthesis is performed in parallel. As a result, an extended DNA chain is generated from many probes P5-2. These are generated as single strands, but probe P5-3 hybridizes to the 3 ′ end to synthesize complementary strands, resulting in double stranded DNA. Heat is applied to form single strands, but the reaction cells are isolated from each other, so the generated floating DNA strands do not exit the reaction cell. Then, it is quickly captured by the probe P5-2 fixed on the cell surface. Subsequent complementary strand synthesis can then be performed to amplify the DNA in the reaction cell. Since each thermal cycle is multiplied by n, a large number of copies of DNA can be produced in the reaction cell in a short time by applying several thermal cycles.

  Although the use of the separated reaction cell is disclosed here, a capillary tube array such as a capillary plate may be used as such a reaction field. In this case, the probe P5-2 is fixed inside the tube. When a solution containing target DNA having a repetitive sequence is passed through a tube array, the target can be efficiently captured with a probe. Subsequent complementary strand synthesis is then performed to produce many copies in the tube. When probe P5-3 is added together, double-stranded DNA is generated in a state of being fixed to the wall. A copy of the target DNA amplified by heat denaturation or the like can be taken out and used.

[Example 6]
In this example, when a probe that hybridizes to the 3 ′ end of a complementary DNA strand that hybridizes to a repetitive sequence portion and synthesizes a complementary strand synthesizes the complementary strand, a portion that includes a recognition site for an enzyme that generates a nick An example to be introduced is disclosed. Using the complementary DNA strand synthesized from primer P6-2 that hybridizes to the repetitive sequence as a template, DNA complementary strand synthesis was performed starting from primer P6-3 that hybridizes to its 3 'end, and the target DNA sequence A DNA strand containing a homologous sequence portion is synthesized. When primer P6-2 synthesizes a complementary strand, an enzyme such as N.BstNBI binds to the recognition site of the cleavage enzyme and cuts only one strand outside the recognition sequence. The recognition sequence of this enzyme is ----- GAGTCT | -----. Of course, other enzymes may be used and are not limited to this. The use of such an enzyme is reported in Anal. Chem. 77, 7984-7972 (2005). Since the above-described strand displacement type polymerase is used as the DNA polymerase, complementary strand synthesis is restarted from the nick portion thus formed. Since this new strand synthesizes the complementary DNA strand while peeling off the already synthesized DNA strand, a single strand of DNA is repeatedly formed. Since there is a repetitive sequence on the 3 ′ end side of this DNA strand, complementary strand synthesis starts and DNA can be amplified repeatedly. This will be described below with reference to the drawings.

  Process (6-1) in FIG. 6 is a target DNA 6-1 having a repeating sequence at the 3 ′ end and a site where the primer P6-3 and a part of the sequence become a priming site of the same sequence at the 5 ′ end. An example using is shown. From the primer P6-2 that hybridizes to the repetitive sequence portion, a DNA strand is generated one after another by the strand displacement type complementary strand synthesis. The resulting DNA strand is released into the reaction solution in a single strand state. As shown in process (6-2), a priming site is formed at the 3 ′ end, and primer P6-3 is hybridized. Primer P6-3 performs complementary strand synthesis using complementary strand DNA6-2 as a template to synthesize DNA6-3 containing the same sequence as target DNA6-1 (process (6-3)). Primer P6-3 introduces a site that is recognized and cleaved by the enzyme when it becomes a double strand, but a double strand is formed by complementary strand synthesis and this site is recognized by the enzyme. As shown in (), a cut (nick) occurs in the middle of the array. Since strand displacement type DNA polymerase can start complementary strand synthesis from this cut, complementary strand synthesis proceeds while peeling the already synthesized DNA strand as shown in processes (6-5) to (6-6). To do. As the complementary strand synthesis proceeds, a nick is formed again and the next complementary strand synthesis is started. In this way, many DNAs having the same sequence can be generated. The generated DNA6-3s has a slightly shorter sequence than DNA6-3 because the 5 'end is a sequence after the nick site (process (6-7)). Since DNA6-3s has a repetitive sequence at the 3 ′ end, primer P6-2 hybridizes to form complementary strand DNA6-2s as shown in processes (6-7) and (6-8). Generate. DNA6-2s hybridizes to primer P6-3 and has the same sequence as DNA6-2, and primer P6-3 creates DNA 6-3 by complementary strand synthesis (process (6-9) and (6-10)). The primer site of DNA6-3 is nicked again and the process from process (6-4) is repeated. Since the process of repeatedly making DNA copies is repeated in this way, a very large number of DNA copies can be generated in a short time.

  In this example, a nick site was inserted into the primer sequence, but this site can also appear only after complementary strand synthesis. In this case, DNA 6-2s synthesized using DNA 6-3s shortened by cleavage as a template does not have a priming site, so primer P6-3 cannot hybridize, and the overall amplification rate slightly decreases.

  It is effective to design primer P6-3 so that it is not cleaved only by hybridizing to the target DNA but is cleaved only after complementary strand synthesis occurs.

  The present invention provides a nucleic acid amplification method. Such a method can amplify any nucleic acid at the same amplification factor even if plural kinds of nucleic acids coexist. In addition, according to such a method, it is possible to amplify a nucleic acid uniformly at a high amplification rate in a short time regardless of the sequence or length of the nucleic acid. Therefore, the amplification method according to the present invention is excellent as a method for uniformly amplifying mRNA, in particular, synthesizing cDNA from a small amount of mRNA obtained from one cell and amplifying the cDNA uniformly without bias. The method according to the present invention is useful in the fields of gene expression analysis, cell function analysis, biological tissue analysis method, disease diagnosis, infectious disease test, drug discovery, etc., where rapid nucleic acid amplification and detection are desired. .

Claims (43)

A method for amplifying nucleic acid, comprising:
Introducing a repetitive sequence containing a known sequence at the 3 ′ end of the target nucleic acid strand containing the target sequence or its complementary sequence;
Hybridizing a plurality of oligonucleotides that hybridize to the repetitive sequence to the repetitive sequence introduced into the target nucleic acid chain;
A method comprising a step of synthesizing complementary strands using the target nucleic acid strand as a template using the oligonucleotide as a primer to generate a plurality of nucleic acid complementary strands from one target nucleic acid strand.   The method according to claim 1, wherein the repetitive sequence is shorter than the length of the target sequence or its complementary sequence.   The method according to claim 1, wherein the repetitive sequence is longer than the length of the target sequence or a complementary sequence thereof.   The method according to any one of claims 1 to 3, wherein the number of repeating units contained in the repeating sequence exceeds 100.   The method according to any one of claims 1 to 3, wherein the number of repeating units contained in the repeating sequence exceeds 1000.   The method according to any one of claims 1 to 5, wherein the target nucleic acid strand comprises a plurality of types of nucleic acid strands, and a common repetitive sequence is introduced into the plurality of types of nucleic acid strands. .   The method according to any one of claims 1 to 6, wherein the introduction of the repetitive sequence is performed by introducing a nucleic acid chain having the repetitive sequence into the target nucleic acid chain by ligation.   For the introduction of repetitive sequences, a nucleic acid chain having a repetitive sequence is used as a template, the 3 ′ end portion of the target nucleic acid strand is used as a primer for repetitive sequence introduction, or the sequence portion added to the 3 ′ end of the target nucleic acid strand is a primer for repetitive sequence introduction The method according to any one of claims 1 to 6, wherein the method is carried out by complementary strand synthesis used as   The method according to claim 7 or 8, wherein the nucleic acid strand having a repetitive sequence is produced by complementary strand synthesis using a ring-shaped nucleic acid as a template.   The introduction of the repetitive sequence is performed by complementary strand synthesis using a sequence portion contained in the target nucleic acid strand or a sequence portion added to the target nucleic acid strand as a primer for repetitive sequence introduction and using a ring-shaped nucleic acid as a template. Item 7. The method according to any one of Items 1 to 6.   Repetitive sequence introduction is performed using an oligonucleotide that can be used to repeat the sequence portion added to the target nucleic acid strand as a primer for repetitive sequence introduction and hybridize to the sequence portion and form a ring-shaped nucleic acid by ligation. The method according to any one of claims 1 to 6, wherein the method is carried out by complementary strand synthesis using the prepared ring-shaped nucleic acid as a template.   The method according to claim 10 or 11, wherein at least a part of the primer for repetitive sequence introduction uses a sequence portion added to the 3 'end of the target nucleic acid strand.   For the introduction of repetitive sequences, a complementary sequence of a sequence portion obtained by complementary strand synthesis using an oligonucleotide that hybridizes to at least a part of the sequence portion added to the 3 'end of the target nucleic acid strand as a template, as a primer for repetitive sequence introduction The method according to any one of claims 1 to 6, wherein the method is performed by complementary strand synthesis using a ring-shaped nucleic acid as a template.   The method according to any one of claims 8 to 13, wherein a sequence moiety is added to the 3 'end of the target nucleic acid strand by terminal transferase.   An oligonucleotide that hybridizes to the sequence portion adjacent to the sequence portion added to the 3 'end of the target nucleic acid strand and to the 3' end side of the target nucleic acid strand and all or part of the added sequence portion and contains a known sequence Is used as a template to synthesize a complementary strand, a part of the sequence added to the 3 ′ end of the target nucleic acid strand is removed as necessary, and then the target nucleic acid is synthesized by complementary strand synthesis using the hybridized oligonucleotide as a template. The repeat sequence is introduced by carrying out complementary strand synthesis using the known sequence added to the 3 ′ end side of the strand as a repeat sequence introduction primer and using a ring nucleic acid as a template. The method described.   The removal of a part of the sequence portion added to the 3 ′ end of the target nucleic acid strand is performed by removing the base in the sequence portion where the oligonucleotide is not hybridized using an enzyme containing exonuclease, Item 16. The method according to Item 15.   The sequence portion used as a repetitive sequence introduction primer added to the 3 ′ end of the target nucleic acid strand is generated by oligonucleotide ligation following restriction enzyme cleavage or complementary strand synthesis using the oligonucleotide as a template. The method according to any one of claims 10 to 13.   The method according to claim 10, wherein a known sequence portion contained in the target nucleic acid chain is used as a primer for repetitive sequence introduction.   The method according to claim 18, wherein a known sequence portion of the target nucleic acid chain is cleaved, and a portion including the 3 'end of the obtained target nucleic acid strand is repeatedly used as a primer for sequence introduction.   The method according to claim 19, wherein the cleavage of the known sequence portion of the target nucleic acid chain is performed by an enzyme containing Cleavase.   Hybridize to a known sequence portion of the target nucleic acid strand and hybridize an oligonucleotide having an anchor sequence on the 5 ′ end side to the target nucleic acid strand, and perform complementary strand synthesis to extend the oligonucleotide to obtain a double-stranded nucleic acid. And then degrading and removing the single-stranded portion of the target nucleic acid strand using an enzyme that degrades the single-stranded nucleic acid from the 3 ′ end, and then the 3 ′ end side of the target nucleic acid strand using the anchor sequence of the oligonucleotide as a template. The method according to any one of claims 10 to 13, wherein a sequence portion is added to the target nucleic acid strand by extending complementary strand synthesis to generate a primer for repeated sequence introduction.   Hybridize to a known sequence portion of the target nucleic acid strand and hybridize an oligonucleotide having an anchor sequence on the 5 ′ end side to the target nucleic acid strand, and perform complementary strand synthesis to extend the oligonucleotide to obtain a double-stranded nucleic acid. Generate and remove the single-stranded portion of the target nucleic acid strand, then add the known sequence portion to the 3 'end of the target nucleic acid strand by complementary strand synthesis using the oligonucleotide anchor sequence as a template, and remove the oligonucleotide The method of any one of Claims 10-13 which produces | generates the primer for repeating arrangement | sequence introduction by doing.   A known sequence portion is added to the 3 ′ end and 5 ′ end of the target nucleic acid strand, and the known sequence portion at the 3 ′ end of both the target nucleic acid strand and its complementary nucleic acid strand is repeatedly used as a primer for sequence introduction. The repeat sequence is introduced into the 3 'end and the 5' end by carrying out complementary strand synthesis using a ring-shaped DNA capable of hybridizing to the template as a template, any one of claims 8 to 11. 2. The method according to item 1.   Complementary strand synthesis is performed using as a primer the first oligonucleotide that hybridizes to the repetitive sequence introduced into the target nucleic acid strand, and the second oligo that hybridizes to the 3 ′ end using the generated complementary nucleic acid strand as a template 24. The method of any one of claims 1 to 23, comprising the step of performing complementary strand synthesis using nucleotides as primers and amplifying a copy of the target nucleic acid strand.   Complementary strand synthesis was performed using an oligonucleotide that hybridizes to a repetitive sequence introduced into the target nucleic acid strand as a primer. The resulting double-stranded nucleic acid was used as a single-stranded nucleic acid, and then the oligonucleotide was used as a primer. The method according to any one of claims 1 to 23, wherein the step of performing complementary strand synthesis using a single-stranded nucleic acid as a template is repeated.   26. The method according to any one of claims 1 to 25, comprising the step of generating a target nucleic acid strand comprising a target sequence or a complementary sequence thereof, having a repetitive sequence at the 3 'end and a loop sequence at the 5' end. the method of.   27. The method according to any one of claims 1 to 26, wherein the nucleic acid strand is a DNA strand.   The method according to any one of claims 1 to 27, wherein the 5 'end side of the target nucleic acid strand is immobilized on a solid surface.   The method according to any one of claims 1 to 28, wherein an oligonucleotide that hybridizes to a repetitive sequence introduced into a target nucleic acid chain is immobilized on a solid surface.   A complementary nucleic acid strand generated by using a first oligonucleotide that hybridizes to a repetitive sequence introduced into a target nucleic acid strand is captured using a second oligonucleotide immobilized on a solid surface, and the second oligonucleotide 30. The method according to any one of claims 1 to 29, comprising a step of performing complementary strand synthesis using as a primer.   31. A method according to any one of claims 28 to 30, wherein the solid is a bead.   31. A method according to any one of claims 28 to 30, wherein the solid is a compartmentalized planar solid.   31. A method according to any one of claims 28 to 30, wherein the solid surface is a porous or inner wall of a pore array.   The method according to any one of claims 29 to 33, wherein at least two kinds of oligonucleotides that hybridize to a target nucleic acid strand or a complementary nucleic acid strand thereof are immobilized on the same solid surface.   A step of synthesizing a complementary strand so that a restriction enzyme cleavage recognition sequence is introduced before or after the repetitive sequence introduced into the target nucleic acid strand, and cleaving the repetitive sequence using the restriction enzyme from the obtained complementary nucleic acid strand. 35. The method of any one of claims 1-34, comprising: Synthesizing a complementary strand using the target nucleic acid strand as a template using a capture probe having a sequence complementary to the target nucleic acid strand and a first anchor sequence at the 5 ′ end;
The generated complementary nucleic acid strand as a single strand,
Complementary strand synthesis is performed using a first oligonucleotide that hybridizes to a single-stranded complementary nucleic acid strand, and a known sequence portion based on the first anchor sequence is introduced into the 3 ′ end of the first oligonucleotide extended. Step,
Using the known sequence portion as a primer to hybridize with a ring-shaped nucleic acid strand to synthesize a complementary strand to generate a complementary nucleic acid strand having the first repeat sequence introduced at the 3 ′ end, and the first repeat The method of claim 1, comprising hybridizing a plurality of second oligonucleotides to the sequence and synthesizing complementary strands in parallel.   The first oligonucleotide contains a second anchor sequence on the 5 ′ end side, and a second oligonucleotide hybridized to the first repeat sequence is used as a primer to synthesize a complementary strand. A known sequence portion based on the anchor sequence of was introduced, a complementary strand synthesis was performed using the known sequence portion as a primer and a ring-shaped nucleic acid as a template, and a second repetitive sequence was introduced on the 3 ′ end side. 40. The method of claim 36, further comprising generating a complementary nucleic acid strand.   The method according to any one of claims 1 to 37, wherein a cDNA library is prepared from a plurality of types of mRNA, and the DNA is uniformly amplified using a plurality of types of cDNA contained in the cDNA library as a target nucleic acid chain.   40. The method of claim 38, wherein the method produces an amplified DNA copy on a bead that is different from the bead that captures the mRNA or cDNA. A method for uniformly amplifying a nucleic acid pool containing single or multiple nucleic acid strands,
Introducing a repetitive sequence containing a known sequence at the 3 ′ end of a nucleic acid strand contained in the nucleic acid pool;
A method comprising a step of performing complementary strand synthesis using an oligonucleotide that hybridizes to the repetitive sequence and a strand displacement polymerase. A method for amplifying nucleic acid, comprising:
Introducing a repetitive sequence containing a known sequence at the 3 ′ end of the target nucleic acid strand containing the target sequence or its complementary sequence;
Performing complementary strand synthesis using an oligonucleotide that hybridizes to the repetitive sequence;
Introducing a cleavage site (nick) into the resulting double-stranded nucleic acid;
A method comprising a step of synthesizing complementary strands from a cleavage portion (nick) to generate a plurality of nucleic acid strands. A method for detecting a nucleic acid in a sample, comprising:
Amplifying nucleic acid in a sample by the method of any one of claims 1-41;
A method comprising a step of detecting a nucleic acid to be detected using the obtained amplification product.   43. The method of claim 42, wherein the method amplifies all nucleic acids in the sample or only the nucleic acid to be detected.

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