JP5223267B2 - Gene amplification product recovery method and gene amplification product recovery kit - Google Patents

Description

  The present invention relates to a method for recovering gene amplification products and a kit for recovering gene amplification products.

  With the development of genetic engineering, gene amplification methods such as the polymerase chain reaction (hereinafter referred to as “PCR reaction”) method are actively used. The polynucleotide amplified by the gene amplification method requires removal of unreacted raw materials and enzymes and buffer replacement depending on the purpose of use of the polynucleotide, such as restriction enzyme reaction and sequencing method.

  As to what kind of treatment is actually performed, it is necessary to appropriately select a treatment method according to the purpose of use of the polynucleotide. One of the processing methods is gel filtration. In gel filtration, the amplified polynucleotide is passed through a column packed with gel. Thereby, the polynucleotide of the target molecular weight can be fractionated. Moreover, the filter column filtration method is known. In the filter column filtration method, the polynucleotide is captured by a filter provided in the column. After the filter is washed, the polynucleotide is re-extracted. For this reason, the removal rate of unreacted substances is high.

  However, the method using filtration requires a large apparatus such as a centrifuge. For this reason, there exists a problem that an apparatus structure becomes large. In addition, there is a problem that a polynucleotide having a small molecular weight that is not captured by a gel or a filter cannot be recovered.

In order to solve such problems, methods other than the method using filtration have been developed. For example, a method of electrostatically adsorbing and recovering a polynucleotide, a method of capturing with a carrier immobilized with streptavidin using a biotinylated primer (for example, see Patent Documents 1 and 2), etc. are known. Yes.
JP 2007-89446 A Special table 2004-500062 gazette

  As described in Patent Documents 1 and 2, the method using a biotinylated primer can isolate the sense strand and / or the antisense strand. However, the biotin-avidin bond is strong. For this reason, the obtained polynucleotide cannot be released / recovered. Therefore, the obtained polynucleotide cannot be used for an enzyme reaction such as a single base extension reaction for detecting a single nucleotide polymorphism.

  In addition, as a method for recovering mRNA, a method using a polyA sequence in a target polynucleotide and a polyT sequence as a recovery polynucleotide is generally known. In this method, the recovery efficiency depends on the hybridization efficiency between the target polynucleotide and the recovery polynucleotide and the biotin-streptavidin binding efficiency. For this reason, there exists a problem that collection | recovery efficiency is very low. In particular, the hybridization efficiency between the target polynucleotide and biotin-labeled polyT is low. For this reason, when the addition amount of biotin-labeled polyT is increased, unreacted biotin-labeled polyT reacts with streptavidin. As a result, the collection efficiency is not improved.

  Further, when a GC hydrogen bond having a stronger binding force than an AT hydrogen bond is used for hybridization between the target polynucleotide and the recovery polynucleotide, the Tm (melting temperature) value increases. For this reason, it becomes difficult to release and recover the target polynucleotide.

  The present invention has been made in view of the above problems, and an object thereof is to provide a method and a recovery kit for selectively and efficiently recovering a gene amplification product.

  As a result of intensive studies to solve the above problems, the present inventors have completed the present invention. In the present invention, first, a target polynucleotide is amplified using a forward primer containing a tag sequence and a reverse primer containing a sequence complementary to the tag sequence, so that the same nucleotide sequence is present at both ends of the single strand. A target polynucleotide comprising the tag sequence is obtained. Thereby, the hybridization efficiency of the target polynucleotide and the recovery polynucleotide can be improved. As a result, the recovery efficiency of the target polynucleotide is improved. That is, the present invention is as follows.

  The gene amplification product recovery method of the present invention comprises a target comprising a first tag sequence having the same base sequence at both ends of the sense strand and a second tag sequence having the same base sequence at both ends of the antisense strand. A first step of amplifying the polynucleotide; and a recovery polynucleotide comprising a base sequence complementary to the first tag sequence and / or the second tag sequence; And / or a second step of fixing via a second tag sequence, wherein the sense strand and / or antisense strand is recovered from the gene amplification product of the double-stranded nucleic acid. .

  After the second step, a third step of releasing and recovering the immobilized target polynucleotide may be included.

  Amplification of the target polynucleotide is performed using a forward primer containing a tag sequence and a reverse primer containing a sequence complementary to the tag sequence.

  The first tag sequence provided at both ends of the sense strand and the second tag sequence provided at both ends of the antisense strand may be the same base sequence or different base sequences.

  The collection polynucleotide may be immobilized on a carrier. In addition, a label may be bound to the recovery polynucleotide, and a substance having specific affinity for the label may be immobilized on the carrier. The carrier is preferably magnetic fine particles. The tag sequence and the recovery polynucleotide may include a non-natural nucleic acid.

The gene amplification product recovery kit of the present invention is a gene amplification product recovery kit comprising a tag sequence that is the same base sequence at both ends in the sense strand and the antisense strand of a target polynucleotide, wherein the tag sequence is comprising comprising a forward primer, a reverse primer comprising a sequence complementary to the tag sequence, and a recovery polynucleotide comprising a phase complementary sequence to said tag sequence. In addition, the recovery polynucleotide may be immobilized on a carrier.

  In the present invention, a target polynucleotide is amplified using a forward primer containing a tag sequence and a reverse primer containing a sequence complementary to the tag sequence, so that the tag sequence has the same base sequence at both ends of a single strand. A target polynucleotide comprising is obtained. A tag sequence having the same base sequence exists at both ends of a single strand. For this reason, the probability of hybridizing with the polynucleotide for collection increases. As a result, the recovery efficiency of the target polynucleotide is improved.

  In the present invention, the target polynucleotide is not captured using a biotin-avidin bond. As a result, the obtained polynucleotide can be easily released and recovered.

In the method for recovering a gene amplification product of the present invention, (1) a first tag sequence having the same base sequence at both ends of a sense strand and two antisense strands using two types of primers containing complementary tag sequences And a step of amplifying a target polynucleotide comprising a second tag sequence having the same base sequence at both ends of the DNA, and (2) a step of immobilizing the amplified target polynucleotide to a recovery polynucleotide. Further, (3) a step of releasing / recovering the immobilized target polynucleotide may be included. The gene amplification product collection kit of the present invention is a kit for reliably performing the above-described steps. Hereinafter, the above steps will be described with reference to the drawings. FIG. 1 is a conceptual diagram illustrating a process of amplifying a target polynucleotide containing a tag sequence having the same base sequence at both ends of a single strand using two types of primers containing complementary tag sequences. FIG. 2 is a process diagram illustrating a process of fixing the amplified target polynucleotide to the recovery polynucleotide and a process of fixing the target polynucleotide to the recovery polynucleotide.

(First step)
As shown in FIG. 1, in the first step, a nucleic acid having a target site 1 is converted into a forward primer 3 having a tag sequence 2 and a reverse primer 5 having a sequence 3 complementary to the tag sequence 2. Perform PCR reaction. The sense strand 6 of the obtained target polynucleotide contains the first tag sequence 2 of “AAAAA” at both ends thereof. On the other hand, the antisense strand 7 includes a second tag sequence 4 of “TTTTTT” complementary to the tag sequence 2 at both ends thereof.

[Target polynucleotide]
The nucleic acid that is the target of the target polynucleotide in the present invention is not particularly limited, and genomic DNA extracted from cells or the like, and those obtained by amplifying a target region using an enzymatic reaction from genomic DNA can be used. When amplifying the target region, the method for amplifying the target region is not particularly limited, and a known amplification method can be used. A preferred amplification method is a PCR reaction using a thermostable DNA polymerase.

  The labeled polynucleotide may be a sense strand, an antisense strand, or a sense strand and an antisense strand obtained from a gene amplification product of a double-stranded nucleic acid. Which of these is used depends on the intended use of the labeled polynucleotide.

[Primer]
The primer used in the present invention may be any primer that can amplify the target region and includes a tag sequence captured by the polynucleotide for recovery. In order to provide a tag sequence that is the same base sequence at both ends of the anti-strand or antisense strand, the tag sequence of the forward primer and the reverse primer must be complementary. In the example of FIG. 1, the forward primer includes the first tag sequence 2 of “AAAAA”. On the other hand, the reverse primer includes a second tag sequence 3 of “TTTTTT” complementary to the first tag sequence 2.

[Tag array]
The tag sequence contained in the primer is not particularly limited as long as it is a sequence complementary to the recovery polynucleotide. Preferably, a sequence that does not mishybridize with a template sequence such as genomic DNA is preferable so as not to cause an error in the PCR reaction. Further, when the target polynucleotide immobilized on the recovery polynucleotide is released / recovered, it is desirable to extract it by substituting with pure water. For this reason, the number of bases that can be dissociated at room temperature and a low salt concentration and the Tm value, that is, the annealing site with the sequence of the target region is 16 to 22 mer (base number) and the Tm value is 55 to 65 ° C.

  The tag sequence may be a non-natural nucleic acid. Here, the non-natural nucleic acid means a nucleic acid that does not exist in nature. Examples of the non-natural nucleic acid include those in which the sugar contained in the nucleic acid is L-form, or is modified. Some non-natural nucleic acids do not hybridize with genomic DNA, which is a natural nucleic acid, or other template DNA. Therefore, when a non-natural nucleic acid is used for the tag sequence, misproliferation in the PCR reaction can be suppressed.

  In the example of FIG. 1, the tag sequence of the sense strand and the tag sequence of the antisense strand are different. Therefore, in the example of this figure, it is effective when only the sense strand or the antisense strand is recovered, or when the sense strand and the antisense strand are recovered with separate recovery polynucleotides. On the other hand, when the sense strand and the antisense strand are recovered with the same recovery polynucleotide, the recovery polynucleotide has a sequence capable of hybridizing with a part of the tag sequence at either the 5 ′ end or the 3 ′ end. do it. For example, the first tag sequence of the sense strand and the second tag sequence of the antisense strand are changed to a sequence including a repetitive sequence such as “... In this case, if the recovery polynucleotide has a sequence of “TATATATATA...”, At least a part of the first tag sequence of the sense strand and the second tag sequence of the antisense strand is at least part of the recovery polynucleotide. It can hybridize with nucleotides. As a result, the sense strand and the antisense strand can be recovered with the same recovery polynucleotide. Thus, in the present specification, the case where the sense strand and the antisense strand have a tag sequence that can be recovered by the same recovery polynucleotide is referred to as “the first strand provided at both ends of the sense strand”. The tag sequence and the second tag sequence provided at both ends of the antisense strand are referred to as “the same base sequence”.

(Second step)
As shown in FIG. 2, the PCR product containing the sense strand / antisense strand complex 8 obtained in the first step is heated to dissociate the sense strand 6 and the antisense strand 7. The PCR product is brought into contact with a collecting polynucleotide 9 containing a base sequence complementary to the tag sequence 2. According to the example of this figure, the sense strand 6 having the first tag sequence “AAAAA” is included in the recovery polynucleotide 9, and hybridizes with “TTTTT” which is a base sequence complementary to this tag sequence. . A label 10 is bound to the recovery polynucleotide 9. In the example of this figure, a substance 12 having a specific affinity for a label is immobilized on the carrier 11. The target polynucleotide is immobilized on the carrier by binding the label and a substance having specific affinity for the label. The release and recovery of the target polynucleotide is performed by washing with pure water, for example.

[Recovery polynucleotide]
The collection polynucleotide is not particularly limited as long as it contains a sequence complementary to the tag sequence of the target polynucleotide and can be immobilized on a carrier. When the tag sequence of the target polynucleotide is a non-natural nucleic acid, it is preferable that the recovery polynucleotide is also a non-natural nucleic acid.

[Immobilization method]
The recovery polynucleotide may be immobilized on a carrier in advance, or may be immobilized on a carrier after hybridizing the labeled polynucleotide. In order to improve the diffusibility of the collection polynucleotide to the PCR product and the contact probability between the target polynucleotide and the collection polynucleotide, the target polynucleotide may be hybridized and then immobilized on a carrier. In particular, a label receptor (a substance having a specific affinity for a label) immobilized on a carrier after the target polynucleotide is hybridized using a label obtained by introducing a label into a collection polynucleotide. And a method of specifically binding to). Specific binding that can be used includes, for example, biotin-avidin reaction, antigen-antibody reaction, chemical binding, ligation reaction and the like. The label may be introduced at either end of the recovery polynucleotide or may be introduced into the sequence. Thus, in the present invention, the method for immobilizing the recovery polynucleotide to the carrier includes the binding strength between the target polynucleotide and the recovery polynucleotide, the binding method, the binding strength between the recovery polynucleotide and the carrier, and the binding method. Use a different one. This facilitates the release and recovery of the target polynucleotide from the recovery polynucleotide.

[Carrier]
The carrier for immobilizing the recovery polynucleotide is not particularly limited as long as non-specific adsorption of DNA is small. For example, particles (metal particles, glass particles, polymer particles, silicon particles, magnetic fine particles, etc.), optical supplement beads, glass slides, resin substrates, paper, gel, nitrocellulose, nylon and the like can be mentioned. These carriers need to be surface-treated to immobilize the polynucleotide for recovery. For example, when the collection polynucleotide is labeled with biotin, the carrier surface is avidinized. When chemically binding using an amino group introduced into the collection polynucleotide, the carrier surface is carboxylated. For example, a functional group such as a group is introduced.

  A preferred carrier is avidin-coated magnetic fine particles. When this carrier is used, since the polynucleotide for recovery can be immobilized in a dispersed state, the immobilization efficiency is excellent. Further, the target polynucleotide can be released and recovered after the magnetic microparticles on which the polynucleotide for recovery has been immobilized are fixed by magnetic force. As a result, it is easy to release and recover the target polynucleotide.

  When immobilized on a carrier, washed with a buffer, and put in a new enzyme reaction solution together with the carrier and used for a labeling reaction, it can be used in the immobilized state.

(Third step)
In the present invention, the target polynucleotide is released and collected as follows. General methods for dissociating DNA double strands include increasing the temperature, decreasing the salt concentration, or a combination thereof. Specifically, the target polynucleotide is released and recovered from the washing solution using the citrate buffer by exchanging the solvent with pure water. Alternatively, the target polynucleotide may be released and recovered by heating to about 50 ° C. in a standard salt concentration solution. For this reason, it is easy to remove excess deoxynucleotide triphosphate and the like generated by the PCR reaction.

  The sense strand and / or the antisense strand recovered by the method of the present invention can be used for analysis of information using the target polynucleotide, such as measuring the number of repeats of a repetitive base sequence in the target polynucleotide.

  The sense strand and / or antisense strand recovered by the method of the present invention can be used for a single-base extension reaction that is an existing enzyme extension reaction. In the single-base extension reaction, excess deoxynucleotide triphosphate existing after the PCR reaction causes a false reaction. In the present invention, for example, the sense strand and / or the antisense strand can be washed using a buffer solution such as a phosphate buffer solution or a citrate buffer solution, so that deoxynucleotide triphosphate can be easily removed. Therefore, it is difficult to cause a false reaction in the single base extension reaction. As a result, SNPs can be detected with high accuracy.

[Detection kit]
The gene amplification product recovery kit of the present invention comprises a forward primer including a tag sequence, a reverse primer including a sequence complementary to the tag sequence, and a recovery poly- gram including a sequence complementary to the tag sequence in the tag sequence. Nucleotides.

  The gene amplification product recovery kit of the present invention includes a forward primer including a tag sequence, a reverse primer including a sequence complementary to the tag sequence, and a recovery including a sequence complementary to the tag sequence in the tag sequence. And a carrier on which the polynucleotide for use is immobilized.

  Alternatively, a forward primer including a tag sequence, a reverse primer including a sequence complementary to the tag sequence, a recovery polynucleotide including a sequence complementary to the tag sequence and a label attached to the tag sequence, It may contain the label and a carrier on which a substance having specific affinity is immobilized.

  The carrier is preferably a magnetic particle. The tag sequence and the recovery polynucleotide may include a non-natural nucleic acid. By using such a gene amplification product recovery kit, the gene amplification product can be selectively and efficiently recovered.

  EXAMPLES Examples will be given below to describe the present invention in detail, but the present invention is not limited to the examples.

Example 1
[Recovery of sense strand using biotin-labeled polyT as a polynucleotide for recovery and use for single base extension reaction]

<A. Preparation of target polynucleotide>
(Primer synthesis)
An annealable amplifiable site was designed, and 25 mer A (adenine) (SEQ ID NO: 1) was placed at the 5 ′ end of the forward primer, and 25 mer T (thymine) (SEQ ID NO: 2) was placed at the 5 ′ end of the reverse primer. The added forward primer and reverse primer were synthesized. The synthesis was outsourced to Operon Biotechnology Co., Ltd.

(Amplification by PCR)
Genomic DNA was extracted using nucleic acid extraction kit “QIAmp” (manufactured by Qiagen) using HEK293 (derived from human fetal kidney cells) cells. The following reagent 1 was added to this genomic DNA and amplified under the following condition 1.

<Reagent 1>
A 50 μl solution containing the following reagents was prepared for each sample.
Forward primer (SEQ ID NO: 1) 15 pmol
Reverse primer (SEQ ID NO: 2) 15 pmol
Buffer 5 μl
2 mM dNTP 5 μl
25 mM MgSO ₄ 2 μl
KOD-plus- DNA polymerase (TOYOBO) 1 U
Extracted DNA solution 50 ng

<Amplification condition 1>
95 ° C · 5 minutes 95 ° C · 30 seconds, 55 ° C · 30 seconds, 68 ° C · 30 seconds (30 cycles)

(Recovery of sense strand)
As the biotin-labeled 25mer polyT, PolyATtract Magnetic mRNA trap kit (Promega) was used. The PCR product was heated at 95 ° C. for 10 minutes, and 3 pmol and 15 pmol of biotin-labeled 25mer polyT were added. This was added to an SSC solution (0.3 M sodium citrate dihydrate, 3 M NaCl solution) containing magnetic fine particles immobilized with streptavidin, and reacted for 10 minutes. This was placed on a magnet and the supernatant was removed. The same operation was performed using a 200-fold diluted SSC solution, and washing was performed three times. The last supernatant was removed as much as possible, 50 μL of pure water was added, and the mixture was extracted for 1 minute with occasional stirring. Magnetic fine particles were immobilized on a magnet, and the supernatant containing the sense strand was recovered.

  Similarly, the supernatant containing the sense strand was recovered using an untagged reverse primer to which 25-mer T (thymine) (SEQ ID NO: 2) was not added to the 5 'end of the reverse primer. Hereinafter, it is shown as “tag sequence only at one end”.

(Measurement of sense strand amount)
In order to measure the amount of DNA in the recovered solution, a nanodrop (manufactured by Perkin Elmer) was used, and the target polynucleotide had a tag sequence at only one end and a recovery polynucleotide in the tag sequence at both ends. Although the concentration was different, the sense strand amount was calculated from the absorbance at 260 nm. The results are shown in Table 2.

  As can be seen from Table 2, the recovery amount of the target nucleotide with the tag sequence added to both ends is larger than when the tag sequence is added to one end or when the addition amount of the collecting polynucleotide is increased. Indicated.

  In addition, the probability of hybridization with the target nucleotide increases as the number of collection polynucleotides increases. On the other hand, the amount of what could not be hybridized also increases. This is considered to be because the binding amount of the recovery polynucleotide hybridized with the target polynucleotide is reduced because it binds to the magnetic fine particles.

<B. Use in single-base extension reactions>
The sense strand recovered by the above method was labeled using a single base extension reaction and detected on a DNA chip.
(Labeling reaction using sense strand)
A 20 μl solution containing the following reagent 2 containing the sense strand recovered by the above method was prepared, and a single base extension reaction was performed under the following condition 2 using this solution. A labeled base was incorporated by the enzymatic method under the following condition 2. The control for Cy3 and the control for Cy5 are polynucleotides for confirming whether Cy3-labeled ddATP and Cy5-labeled ddUTP are normally incorporated, respectively. A complementary base chain with a tag sequence for Cy3 and a complementary base chain with a tag sequence for Cy5 bind to a control for Cy3 and a control for Cy5, and are subjected to a single base extension reaction using the control for Cy3 and the control for Cy5 as a template, respectively. This is a polynucleotide for labeling the ends of the complementary base chain with tag sequence for tag and the complementary base chain with tag sequence for Cy5 and confirming that the reaction proceeds normally. The base sequences of the complementary base chain with tag sequence, Cy3 control, Cy3 tag sequence complementary base chain, Cy3 control and Cy5 tag sequence complementary base chain are shown in SEQ ID NO: 3 in Table 3, respectively. 7 is a base sequence corresponding to 7.

＜反応条件２＞
９５℃・２分
９５℃・２０秒、６０℃・１５秒（３０サイクル）
９５℃・５分
０℃・３分 <Reagent 2>
Sense strand recovery (target polynucleotide) 100 ng
Complementary base chain with tag sequence for signal intensity detection (SEQ ID NO: 3) 20.3 pmol
10 μM Cy3-labeled ddATP 1 μl
10 μM Cy5 labeled ddUTP 1 μl
10 μM ddGTP 1 μl
10 μM ddCTP 1 μl
Buffer 2 μl
Thermo sequence polymerase 8 U
Cy3 control (SEQ ID NO: 4) 12.5 ng
Complementary base chain with tag sequence for Cy3 (SEQ ID NO: 5) 20.3 pmol
Control for Cy5 (SEQ ID NO: 6) 12.5 ng
Complementary base chain with tag sequence for Cy5 (SEQ ID NO: 7) 20.3 pmol

<Reaction condition 2>
95 ° C, 2 minutes, 95 ° C, 20 seconds, 60 ° C, 15 seconds (30 cycles)
95 ° C, 5 minutes 0 ° C, 3 minutes

(Creation of DNA chip)
A polymethyl methacrylate (PMMA) substrate (Cosmo glass extruded plate, thickness: 1 mm, manufactured by Kuraray Co., Ltd.) was immersed in a 10N sodium hydroxide solution at a temperature of 65 ° C. for 12 hours. The PMMA substrate was washed with pure water, 0.1N hydrochloric acid aqueous solution, and pure water in this order.

  By synthesizing three types of signal intensity detection tag sequences D-type nucleic acid complementary strands designed so that there is no cross-hybridization between the signal intensity detection tag sequences and the 5 ′ end aminated, did.

  Each of the synthesized complementary strand DNAs of the three signal intensity detection tag sequences was dissolved in pure water at a concentration of 0.3 nmol / μL, and then 0.03 nmol / μL using a phosphate buffer (pH 5.5). Dilute to Further, in order to condense the carboxylic acid on the substrate surface with the terminal amino group of the immobilized DNA, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) is added so that the final concentration is 50 mg / mL. added.

  The mixed solution was spotted on the upper surface of the PMMA substrate treated with NaOH using an arrayer (manufactured by Nippon Laser Electronics Co., Ltd .: Gene Stamp-II). The number of spots was 16 points, 4 points each for the complementary strand DNA of each tag sequence. The substrate was then placed in a sealed plastic container and incubated at 37 ° C. for 20 hours. Thereafter, the substrate was washed with pure water to produce a DNA chip.

(Preparation of hybridization solution)
Hybridization of the labeled polynucleotide and the immobilized probe for hybridization was performed as follows. The 15 × SSC used below means 20 × SSC (0.3 M sodium citrate dihydrate, 3 M NaCl solution: manufactured by Sigma) diluted to 3/4 with pure water.

  An aqueous solution containing 30 mg / mL BSA (bovine serum albumin), 15 × SSC, 0.3 wt% SDS (sodium dodecyl sulfate), 0.01 wt% salmon sperm DNA (each of the above concentrations is a final concentration) was prepared. . This and the labeling solution prepared according to the above reaction condition 2 were mixed at a ratio of 3: 1 to prepare the same total volume of 40 μL.

(Immobilization to immobilization carrier)
40 μL of the hybridization solution was placed on the substrate carrying the immobilization carrier, covered with a cover glass, incubated at 42 ° C. for 4 hours, and then the cover glass was peeled off, washed and dried.

(Measurement of labeling amount)
The substrate was set on a DNA chip scanner (Perkin Elmer, Scan Array Express), and the signal intensity was measured. The measurement conditions were adjusted to a laser output of 90% and a photomultiplier sensitivity of 60%. Here, the signal intensity refers to an average value of fluorescence intensity in each spot. The results are shown in Table 4. In Table 4, the signal strength of Cy5 in Cy3 control and the signal strength of Cy3 in Cy5 control correspond to noise.

  From Table 4, it can be seen that the signal intensity of Cy5 in Cy3 control and the signal intensity of Cy3 in Cy5 control are very weak. That is, it can be seen that there is no noise due to a false reaction in the single base extension reaction with the PCR product obtained by this recovery method. From this, it was shown that a sufficient dNTP was removed and a sample suitable for a single base extension reaction could be provided.

(Example 2)
[Recovery of sense strand and antisense strand using non-natural nucleic acid as tag sequence]
(Primer synthesis)
An annealing site capable of amplifying the target polynucleotide is designed, a tag sequence using an L-type nucleic acid containing a 23-mer AT repeat at the 5 ′ end of the forward primer and the reverse primer is synthesized, and the annealed portion uses a D-type nucleic acid. Polynucleotides were synthesized. In addition, as a polynucleotide for recovery, a 24-mer AT repeat (SEQ ID NO: 8) shown in Table 5 and labeled with biotin at its 5 ′ end was synthesized with an L-type nucleic acid. The synthesis was outsourced to Operon Biotechnology Co., Ltd. As the L-type nucleic acid, Beta-L-deoxy Adenosine (n-bz) CED phosphoramidite, Beta-L-deoxy Thmidine CED phosphoramidite (manufactured by ChemGenes Corporation) was used.

(Amplification by PCR)
The HEK293 cell genomic DNA described in Example 1 was used, and the following reagent 3 was added, followed by amplification under the above reaction condition 2.

<Reagent 3>
50 μl solutions containing the following reagents were prepared for each sample.
Forward primer (SEQ ID NO: 9) 15 pmol
Reverse primer (SEQ ID NO: 10) 15 pmol
Buffer 5 μl
2 mM dNTP 5 μl
25 mM MgSO ₄ 2 μl
KOD-plus-DNA polymerase (TOYOBO) 1 U
Extracted DNA solution 50 ng

(Recovery of sense strand and antisense strand)
3 pmol of biotin-labeled 24mer polyAT, which is a polynucleotide for collection, was added to the PCR product. Using this, the PCR product was recovered in the same manner as in the method described in Example 1. Further, in the same manner as in Example 1, PCR products were collected using a reverse primer without a tag (a tag sequence only at one end).

(Measurement of PCR product amount)
In order to measure the amount of DNA in the collected solution, it was calculated from the absorbance at 260 nm using Nanodrop (manufactured by Perkin Elmer). The results are shown in Table 6.

  Table 6 shows that in this experiment as well, the PCR product having the tag sequence for both primers had a higher yield than the PCR product having the tag sequence only at one end.

  The PCR product was evaluated by electrophoresis. Electrophoresis was performed by running a 2% agarose gel at 100 V for 30 minutes using a developing solution in which 20 × TAE Buffer (manufactured by Invitrogen) was diluted 20 times. After electrophoresis, the nucleic acid was stained with an ethidium bromide aqueous solution, and the nucleic acid fragment was detected under ultraviolet irradiation at 280 nm.

Ethidium bromide is a reagent that cannot stain single-stranded nucleic acids but stains only double-stranded nucleic acids. Since the PCR product was stained with this reagent, it was confirmed that the sense strand and the antisense strand were recovered.

FIG. 1 is a conceptual diagram illustrating a process of amplifying a target polynucleotide containing a tag sequence having the same base sequence at both ends of a single strand using two types of primers containing complementary tag sequences. FIG. 2 is a process diagram illustrating a process of fixing the amplified target polynucleotide to the recovery polynucleotide and a process of fixing the target polynucleotide to the recovery polynucleotide.

Explanation of symbols

1 target site 2 first tag sequence 3 forward primer 4 second tag sequence 5 reverse primer 6 sense strand 7 antisense strand 8 sense strand / antisense strand complex 9 recovery polynucleotide 10 label 11 carrier 12 label and specific Affinity Substance 13 Target Polynucleotide (Recovered Sense Strand 6)

Claims (9)

A first step of amplifying a target polynucleotide comprising a first tag sequence having the same base sequence at both ends of the sense strand and a second tag sequence having the same base sequence at both ends of the antisense strand;
The target polynucleotide is passed through the first tag sequence and / or the second tag sequence to a recovery polynucleotide comprising a base sequence complementary to the first tag sequence and / or the second tag sequence. A method for recovering a gene amplification product, comprising recovering a sense strand and / or an antisense strand from a gene amplification product of a double-stranded nucleic acid, comprising a second step of fixing. After the second step,
The method for recovering a gene amplification product according to claim 1, comprising a third step of releasing and recovering the immobilized target polynucleotide.   The method for recovering a gene amplification product according to claim 1 or 2, wherein the target polynucleotide is amplified using a forward primer including a tag sequence and a reverse primer including a sequence complementary to the tag sequence.   The first tag sequence provided at both ends of the sense strand and the second tag sequence provided at both ends of the antisense strand are the same base sequence or different base sequences. A method for recovering the gene amplification product according to claim 1.   The method for recovering a gene amplification product according to any one of claims 1 to 4, wherein the polynucleotide for recovery is immobilized on a carrier.   The gene amplification according to any one of claims 1 to 4, wherein a label is bound to the recovery polynucleotide, and a substance having specific affinity for the label is immobilized on the carrier. Product recovery method. A kit for recovering a gene amplification product comprising a tag sequence that is the same base sequence at both ends of each of a sense strand and an antisense strand of a target polynucleotide,
A forward primer comprising the tag sequence;
And a reverse primer comprising a sequence complementary to the tag sequence,
And a recovery polynucleotide comprising a phase complementary sequence to said tag array, collection set of gene amplification products. A kit for recovering a gene amplification product comprising a tag sequence that is the same base sequence at both ends of each of a sense strand and an antisense strand of a target polynucleotide,
A forward primer comprising the tag sequence;
And a reverse primer comprising a sequence complementary to the tag sequence,
And a carrier recovery polynucleotide comprising a phase complementary sequences in the tag sequence is immobilized, collection set of gene amplification products. A kit for recovering a gene amplification product comprising a tag sequence that is the same base sequence at both ends of each of a sense strand and an antisense strand of a target polynucleotide,
A forward primer comprising the tag sequence;
And a reverse primer comprising a sequence complementary to the tag sequence,
And recovering the polynucleotide the tag sequence includes a phase complementary sequences, and to bind the labeled,
A gene amplification product recovery kit comprising the label and a carrier on which a substance having specific affinity is immobilized.

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