CN106754904A - A kind of specific molecular label of cDNA and its application - Google Patents
A kind of specific molecular label of cDNA and its application Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
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- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Abstract
A kind of specific molecular label of cDNA and its application, the specific molecular label include：Universal primer sequence as shown in SEQ ID NO.1；Transposase joint sequence P7 as shown in SEQ ID NO.2；Random sequence：(N)n, N is random sequence, common n；And have 30 poly of T alkali (T) sequences, specific nucleotide sequence such as SEQ ID NO.3 institutes.Specific molecular label is applied into cDNA to build in storehouse, in RNA reverse transcriptions into during full-length cDNA, the specific molecular label is added in the 5` ends of each cDNA, each gene is set to be that unique, follow-up information analysis can be according to each gene in the sequence of special molecular label quantization sample.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of specific molecular label of cDNA and its application.
Background technology
In past twenty or thirty year, the extensive gene sequence analysis of two generations sequencing have greatly promoted genome and have turned
The sequence analysis of record group.The sequencing of transcript profile to whole transcript profile because need not be pre-designed probe and can detect unknown
Transcript, has gradually replaced traditional chip gene expression profile to turn into first-selected transcriptome analysis scheme.
Transcript profile refers in a certain moment, by cell transcription whole RNA out, including mRNA and non-coding RNA.
Transcription group is exactly that the expression of gene is studied from rna level, the situation of genetic transcription in studying cell in integral level
And the rule of transcriptional control.Different from genome, transcription group research contains the restriction in time and space, and same cell is not
Under same growth period and growing environment, its expression conditions is incomplete same.Traditional transcriptome analysis need number
Substantial amounts of gene expression is averaged and is analyzed by ten thousand to hundreds thousand of cells, this method based on population level, it is impossible to
The heterogeneity of gene expression between announcement cell.In addition, when target cell number is rare and is difficult to cultivate, this traditional divides
Analysis method is just helpless, it is necessary to use unicellular transcriptome analysis technology.
The resolution ratio of unicellular transcript profile sequencing technologies is accurate to individual cells, to distinguish various cells in heterogeneous population
The transcript profile feature of type provides strong instrument.Traditional banking process uses the method fragmentations such as ultrasonic wave, atomization
DNA is, it is necessary to larger amount of parent material, is then repaired by end, adds the realization of the steps such as joint.But based on single celled
The initial total serum IgE amount of transcript profile sequencing is few, and this method is not suitable for, and this is to allow many potential users to bring down a bit.
Construction cDNA library is the key technology step of unicellular transcriptome analysis, and its method includes that separation is unicellular, thin
Cellular lysate, mRNA reverse transcriptions into cDNA, cDNA fragmentation and adjunction head, the several steps of amplified library.Single celled transcript profile is surveyed
Sequence starting total serum IgE amount is few, and poly thymidine (oligo-dT) is used in the case where rRNA is not removed as reverse
Record primer obtains substantial amounts of full-length cDNA, using template switch (template-switching) activity of reverse transcriptase in cDNA
3 ｀ ends add the preceding paragraph joint sequence, this section of joint sequence to can be used for full-length cDNA amplification and then obtain the sequence of whole mRNA,
So as to avoid the Preference at 3 ｀ ends.Can be added at fragmentation DNA two ends while interrupting using the banking process of transposase
Joint, the steps such as cumbersome DNA fragmentation, end reparation and coupled reaction are become for the simple enzymatic reaction of a step, this
Method greatly reduces starting template amount and sample processing time.
The research of transcript profile can study gene function and gene structure from integral level, disclose dividing for bioprocess
Handset is managed, and quantifies the expression change wherein of each transcript, thus be widely used in biological study, medicament research and development,
The field such as basic research and clinical diagnosis.However, determining the absolute number of different genes in two different plant species or simple sample
The amount very challenging property of abundance, due to the Preference of PCR amplifications, the mRNA of variable expression is expanded in the PCR that storehouse is continued after
The information for reflecting afterwards has an inaccuracy, and follow-up analysis of biological information data cannot real response gene expression quantity.
The content of the invention
It is specific molecular label and its application for providing a kind of cDNA, the specific molecular mark that the purpose of the present invention is
Label are added in the 5 ｀ ends of each cDNA, make each gene be that unique, follow-up information analysis can be according to the spy
The sequence of opposite molecule label quantifies each gene in sample.
To reach above-mentioned purpose, technical scheme is as follows：
A kind of specific molecular label of cDNA, includes in its full length sequence：Universal primer sequence；Transposase joint sequence
P7；Random sequence：(N)n, N is random sequence, common n；With poly (T) sequence；Wherein, the universal primer sequence such as SEQ
Shown in ID NO.1, as shown in SEQ ID NO.2, poly (T) sequence has 30 i.e. T30VN of T bases to transposase joint sequence.
Preferably, the specific molecular label of cDNA of the present invention, its nucleotide sequence as shown in SEQ ID NO.3,
Particular sequence is as follows：AAGCAGTGGTATCAACGCAGAGTACGTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG(N)nT30VN, wherein, N is random sequence, common n, and N is selected from A, T, C, G；Preferably, n is 5-10.
Application of the specific molecular label of cDNA of the present invention in cDNA builds storehouse.The specific molecular of the cDNA
The cDNA that label is also applied to be interrupted using transposase is built in storehouse.
The present invention also provides the cDNA banking process that a kind of utilization transposase is interrupted, and it comprises the following steps：
1) separation is unicellular, will discharge total serum IgE after slender cellular lysate；The spy with mRNA as template, described in claim 1 or 2
Opposite molecule label is that primer reverse transcription synthesizes the first chain cDNA, the specific molecular label is added in the 5 ' of the first chain cDNA
End, and in process of reverse-transcription, using the template switch activity of reverse transcriptase in the 3 ' ends of the first chain cDNA plus the preceding paragraph joint
Sequence, the joint sequence is identical with the universal primer sequence as shown in SEQ ID NO.1；It is template with the first chain cDNA, with such as
Universal primer sequence shown in SEQ ID NO.1 expands the full-length cDNA of anamorphic zone specific molecular label for primer by PCR,
And pcr amplification product is purified；
2) by step 1) gained full-length cDNA and transposase embedding compound is incubated, completion cDNA fragmentations and adjunction
Head, wherein transposase embedding compound include transposase and annealing joint；
3) using sense primer N7 and anti-sense primer N5 to step 2) fragmentation products that obtain enter performing PCR amplification；Wherein,
The sequence of sense primer N7 is as shown in SEQ ID NO.4：CAAGCAGAAGACGGCATACGAGATGTCTCGTGGGCTCGG, under
The sequence of trip primer N5 is as shown in SEQ ID NO.5：AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTC；
4) to step 3) gained pcr amplification product purify, obtain the cDNA with specific molecular label 5` ends text
Storehouse.
Further, step 2) in, each method processed of the transposase embedding compound includes：Preparation system：Preparation system：
Transposase (10U) 12-15 μ L, annealing joint 5 μ L, embedding buffer solution 12-15 μ L, the μ L of cumulative volume 33, are gently blown and beaten with pipettor
Mix, the reaction system that will be prepared is placed in PCR instrument 30 DEG C and reacts 1.5 hours, -20 DEG C of preservations.
Transposase in the transposase embedding compound is Tn5 families transposase, wild type Tn5 transposases, saltant type
Tn5 transposases are applied to the present invention；It is described annealing joint nucleotide sequence as shown in SEQ ID NO.6, specially：
CTCTTATACACATCTGACGCTGCCGACGA。
Again, step 2) in, described cDNA fragmentations and plus jointing method include：Prepare reaction mixture：The swivel base
Enzyme embeds compound 3-7 μ l, swivel base enzyme reaction buffer solution 4-8 μ l；By 5` ends plus the specific molecular label cDNA with
Reaction mixture mixes, and is blown and beaten with pipettor and mixed；55 DEG C of reaction 10-15min in PCR instrument.
Specific molecular label of the present invention has 4nKind, by 30 T bases of poly thereon (T) sequence with
Poly (A) tail Annealing complementary on mRNA carries out reverse transcription in the presence of reverse transcriptase and obtains the first chain cDNA, specificity point
Subtab is added in the 5 ' ends of the first chain cDNA, and one is added at the 3 ' ends of the first chain cDNA using the template switch activity of reverse transcriptase
Section and universal primer sequence identical joint sequence；Obtained by the amplifiable synthesis of the universal primer as shown in SEQ ID NO.1
Full-length cDNA with specific molecular label, the full-length cDNA is without 3` and 5` Preferences.
The banking process that use transposase of the present invention is interrupted, in cDNA fragmentation process, fragmentation fracture
Two ends are coupled with the annealing joint of transposase, i.e., in addition to 3` the and 5` ends of cDNA, the two ends of the DNA of fragmentation have identical to move back
Fiery joint sequence P5 (as shown in SEQ ID NO.6)；And transposase joint sequence is carried on the 5` end molecular labels of cDNA
Row P7, therefore using the N7 primers with transposase joint sequence (as shown in SEQ ID NO.2) and with annealing joint sequence
The 5` ends of enrichment cDNA can be reacted if the N5 primers of (as shown in SEQID NO.6) by PCR.Follow-up analysis of biological information
Can distinguish each in library by the random sequence in the specific molecular label that is marked on the 5` ends of each cDNA
The source of individual cDNA, realizes the absolute quantitation of cDNA.
In full-length cDNA preparation process, specific molecular label of the present invention is added in the 5 ' ends of the first chain cDNA, from
And stamped a random molecular label to each mRNA, make each gene be unique, follow-up
Even if having different degrees of Preference in the enrichment process of cDNA 5` ends, but for the cDNA of identical molecular label, can be designated as coming
Same copy is come from, therefore just can be with distinguishing different by the different of molecular label during biological data analysis
MRNA, so as to the copy number to mRNA accurately quantify.
Beneficial effects of the present invention：
(1) compared with existing cDNA builds the sequence commonly used in storehouse, the molecular label of present invention design has 4 different sequences
Part constitutes, and this 4 parts play different effects respectively：Universal primer sequence：Due to reverse transcriptase in process of reverse-transcription
Template switch activity adds the preceding paragraph and universal primer identical sequence at the 3` ends of cDNA, synthesizes by the way that universal primer is amplifiable
To the full-length cDNA with specific molecular label；Transposase joint sequence P7：On the 5` end molecular labels of full-length cDNA
With transposase joint sequence P7, and in cDNA fragmentation process, the two ends of the DNA of fragmentation have identical annealing to connect
Header sequence P5, is connect using the sense primer N7 with transposase joint sequence P7 (as shown in SEQ ID NO.2) and with annealing
The 5` ends for being only enriched with cDNA can be reacted by PCR if the anti-sense primer N5 of header sequence P5 (as shown in SEQ ID NO.6)；At random
Sequence：Mark each mRNA molecule；Poly (T) sequence：With 3` ends poly (A) sequence complementary pairing of mRNA, in reverse transcription
Reverse transcription synthesizes the first chain cDNA in the presence of enzyme.
(2) compared with existing transposase interrupts banking process, cDNA of the invention builds transposase embedding used in storehouse
Joint sequence in compound only has a kind of annealing joint sequence P5, by the sense primer N7 with transposase joint sequence P7
It is all annealing joint sequence P5 that two ends can be abandoned during PCR with the anti-sense primer N5 with annealing joint sequence P5
DNA fragmentation and be only enriched with the 5` ends of cDNA.The sequence that follow-up information analysis only needs to the 5` ends of cDNA just can know that it is represented
MRNA, the cDNA sequence information without obtaining total length.
(3) molecular label provided by the present invention is added in the 5` ends of each cDNA, each gene is had an only nothing
Two molecular label, follow-up information analysis can be according to each gene in the sequence of label quantization sample, and accuracy is significantly
Improve.
(4) when the specific molecular label designed by the present invention is applied to construction cDNA library, required initial total amount RNA is low
(1-1000 cell, 10pg-10ng), individual cells can be used as starting template and carry out efficient amplification, solve rare sample
Measure not enough problem；Greatly reduce the treatment of RNA samples simultaneously, so as to avoid the loss of sample, improve operation into
Power.
Specific embodiment
Technical scheme is described in further detail below in conjunction with specific embodiment.
Embodiment 1 designs the specific molecular label of cDNA
The specific molecular label of cDNA is included：
A, universal primer sequence, as shown in SEQ ID NO.1, for subsequent PCR amplification full-length cDNA, universal primer is specific
Sequence is as follows：AAGCAGTGGTATCAACGCAGAGTAC；
B, transposase joint sequence P7, as shown in SEQ ID NO.2, particular sequence is as follows：GTCTCGTGGGCTCGG
AGATGTGTATAAGAGACAG；
C, random sequence (N)n, wherein, N is random sequence, common n；
D, poly (T) sequence, totally 30 T bases are T30VN；
CDNA molecular labels are synthesized by method for synthesizing gene, its nucleotide sequence is as shown in SEQ ID NO.3：
AAGCAGTGGTATCAACGCAGAGTAC GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG(N)nT30VN, wherein, N
It is random sequence, common n, N is selected from A, T, C, G；Preferably, n is 5-10.
Embodiment 2 prepares the full-length cDNA with the specific molecular label
293 cells for selecting people are experiment material, and the Discover-sc WTA Kit of (Vazyme) are only praised using Nanjing promise
V2 (N711) kit carries out transcript profile amplification.Used reagent and material is provided by the kit, for example, unicellular split
Solution liquid, RNase Inhibitor, RT Buffer, DTT, TSOligo, reverse transcriptase, high-fidelity enzyme etc..
(1) preparation of samples
1) 10 × Sample Buffer are prepared in the following order, is gently mixed with pipettor, and of short duration be collected by centrifugation.
Table 1
| Component | Volume |
| Unicellular lysate | 19μl |
| RNase Inhibitor | 1μl |
| Cumulative volume | 20μl |
2) in the PCR pipe of 10 × Sample Buffer to the 0.2ml for taking 1 μ l, the nuclease free pure water of 4 μ l is added
(Nuclease-free H2O)。
3) single celled separation
293 cell suspending liquids of fresh cultured are taken, is collected by centrifugation at normal temperatures, abandoned nutrient solution plus PBS is resuspended, it is aobvious being inverted
Single celled selection is carried out under micro mirror to separate, and unicellular is added to step 2 by isolated) containing 10 × Sample
In the PCR pipe of the 0.2ml of Buffer, tube wall mixing is flicked, be incubated at room temperature 5min.
(2) first chain cDNA synthesize
1) sample after cracking is placed on ice, prepares reaction mixture according to table 2, then 4 μ l reaction mixtures are added
To in 6 μ l cracked unicellular sample, gently mixed with pipettor, it is of short duration be collected by centrifugation after be placed on ice.
Table 2
| Component | Volume |
| Special molecular label as shown in SEQ ID NO.3 | 2μl |
| dNTP | 2μl |
| Cumulative volume | 4μl |
2) 72 DEG C of reaction 3min in PCR instrument, are immediately placed on 2min on ice after terminating.
3) by PCR instrument be preheated to 42 DEG C it is standby.
4) according to table 3 prepare reaction mixture, gently mixed with pipettor, it is of short duration be collected by centrifugation after be placed on ice.
Table 3
| Component | Volume |
| Nuclease-free H2O | 2.5μl |
| Step 2) product | 10μl |
| RT Buffer | 4μl |
| DTT | 1μl |
| RNase Inhibitor | 0.5μl |
| TS Oligo | 1μl |
| Reverse transcriptase | 1μl |
| Cumulative volume | 20μl |
5) PCR reactions as shown in table 4 are carried out, be placed in product on ice after terminating by reaction, and product synthesizes for the first chain cDNA
Product.
Table 4
| Heat lid | 105℃ |
| 42℃ | 90min |
| 70℃ | 15min |
| 4℃ | Maintain |
This step reverse transcription has synthesized the first chain cDNA, and identical with universal primer sequence at the 3 ' ends of cDNA plus the preceding paragraph
Joint sequence.
(3) full-length cDNA amplification with molecular label
It is that primer expands synthesis full-length cDNA by PCR by the universal primer sequence as shown in SEQ ID NO.1, specifically
Step is as follows：
1) PCR reaction solutions as shown in table 5 are configured, are gently mixed with pipettor, it is of short duration be collected by centrifugation after be placed on ice.
Table 5
2) program as shown in table 6 is run in PCR instrument：
Table 6
(4) PCR primer purifying
VAHTS DNA Clean Beads (N411) for only praising (Vazyme) using Nanjing promise adsorbs PCR primer, 80%
Ethanol cleans magnetic bead, and Elution Buffer wash-outs, resulting product is the full-length cDNA with specific molecular label.
Embodiment 3 uses the DNA banking process of transposase
(Vazyme) TruePrep DNA Library Prep produced using Nanjing Vazyme Biotechnology Co., Ltd.
Kit V2 for Illumina (TD503) kit is carried out, and used reagent and material is provided by the kit, for example,
Swivel base enzyme buffer liquid, PCR buffer solutions, high-fidelity enzyme etc..
(Vazyme) Tn5 that transposase and embedding buffer solution are produced by Nanjing Vazyme Biotechnology Co., Ltd.
Transposome (S111) kit is provided.
(1) joint embedding
Configuration reaction system as shown in table 7, mixing is gently blown and beaten with pipettor, and the reaction system that will be prepared is placed in PCR
30 DEG C are reacted 1.5 hours on instrument, obtain transposase embedding compound, -20 DEG C of preservations.
Table 7
(2) the full-length cDNA fragmentation with molecular label
1) add each reactive component as shown in table 8 successively in the PCR pipe that sterilizes, mixing is gently blown and beaten using pipettor.
Table 8
| Component | Volume |
| 1ng cDNA | 1μl |
| Swivel base enzyme buffer liquid | 4μl |
| Transposase embeds compound | 5μl |
| ddH2O | 40μl |
| Total | 50μl |
2) PCR reactions as shown in table 9 are carried out, immediately to 5 × TS that 5 μ l are added in product, using pipettor gently
Piping and druming is mixed, and room temperature places 5min.
Table 9
| Heat lid | 105℃ |
| 55℃ | 10min |
| 10℃ | Hold |
(3) 5 ' ends of PCR enrichments cDNA
1) PCR reaction solutions as shown in table 10 are configured, are gently mixed with pipettor, it is of short duration be collected by centrifugation after be placed on ice.
Table 10
Wherein, the sequence of sense primer N7 is as shown in SEQ ID NO.4：CAAGCAGAAGACGGCATACGAGATxxxxxx
XxGTCTCGTGGGCTCGG, wherein xxxxxxxx are index sequences；The sequence of anti-sense primer N5 is as shown in SEQ ID NO.5：
AATGATACGGCGACCACCGAGATCTACACxxxxxxxxTCGTCGGCAGCGTC, xxxxxxxx are index sequences.
2) program as shown in table 11 is run in PCR instrument：
Table 11
(3) PCR primer purifying
The VAHTS DNA CleanBeads that (Vazyme) bio tech ltd produces only are praised using Nanjing promise
(N411) PCR primer is adsorbed, 80% ethanol cleaning magnetic bead, Elution Buffer wash-outs, the product for finally giving is richness
Collect the library at the 5 ' ends of cDNA.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although ginseng
The present invention has been described in detail according to preferred embodiment, it will be understood by those within the art that, can be to invention
Technical scheme is modified or equivalent, and without deviating from the spirit and scope of technical solution of the present invention, it all should cover
In scope of the presently claimed invention.
Sequence table
<110>Nanjing Vazyme Biotechnology Co., Ltd.
<120>A kind of specific molecular label of cDNA and its application
<160> 6
<210>SEQ ID NO. 1
<211>25
<212>DNA
<213>It is artificial synthesized
AAGCAGTGGTATCAACGCAGAGTAC
<210>SEQ ID NO. 2
<211>34
<212>DNA
<213>It is artificial synthesized
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
<210>SEQ ID NO. 3
<211>61
<212>DNA
<213>It is artificial synthesized
AAGCAGTGGTATACGCAGAGTACGTTCGTGGGCTCGGTTCATAAGAGACAGNNNNNNNT30VN
<210>SEQ ID NO. 4
<211>39
<212>DNA
<213>It is artificial synthesized
CAAGCAGAAGACGGCATACGAGATGTCTCGTGGGCTCGG
<210>SEQ ID NO. 5
<211>43
<212>DNA
<213>It is artificial synthesized
AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTC
<210>SEQ ID NO. 6
<211>29
<212>DNA
<213>It is artificial synthesized
CTCTTATACACATCTGACGCTGCCGACGA
Claims (7)
1. a kind of specific molecular label of cDNA, includes in its full length sequence：Universal primer sequence；Transposase joint sequence
P7；Random sequence：(N)n, N is random sequence, common n；With poly (T) sequence；Wherein, the universal primer sequence such as SEQ
Shown in ID NO.1, as shown in SEQ ID NO.2, poly (T) sequence has 30 T alkali to the transposase joint sequence P7
Base is T30VN.
2. the specific molecular label of cDNA according to claim 1, it is characterised in that the core of the specific molecular label
Nucleotide sequence is as shown in SEQ ID NO.3, and particular sequence is as follows：AAGCAGTGGTATCAACGCAGAGTACGTCTCGTGGGCTC
GGAGATGTGTATAAGAGACAG(N)nT30VN, wherein, N is random sequence, and common n, n is 5-10.
3. application of the specific molecular label of cDNA as claimed in claim 1 or 2 in cDNA builds storehouse.
4. the specific molecular label of cDNA as claimed in claim 1 or 2 in the cDNA interrupted using transposase builds storehouse should
With.
5. the cDNA banking process that a kind of utilization transposase is interrupted, it is characterised in that comprise the following steps：
1) separation is unicellular, will discharge total serum IgE after slender cellular lysate；The specificity with mRNA as template, described in claim 1 or 2
Molecular label is that primer reverse transcription synthesizes the first chain cDNA, and the specific molecular label is added in the 5 ' ends of the first chain cDNA, and
In process of reverse-transcription, using the template switch activity of reverse transcriptase in the 3 ' ends of the first chain cDNA plus the preceding paragraph joint sequence,
The joint sequence is identical with the universal primer sequence as shown in SEQ ID NO.1；It is template with the first chain cDNA, with such as SEQ ID
Universal primer sequence shown in NO.1 expands the full-length cDNA of specific molecular label described in anamorphic zone for primer by PCR, and
Purified；
2) by step 1) gained full-length cDNA and transposase embedding compound is incubated, completion cDNA fragmentations and adjunction head；
Wherein, the transposase embedding compound includes transposase and annealing joint；
3) using sense primer N7 and anti-sense primer N5 to step 2) obtained by fragmentation products enter performing PCR amplification, be enriched with cDNA
5 ' end；Wherein, the sequence of sense primer N7 is as shown in SEQ ID NO.4, the sequence such as SEQ ID NO.5 institutes of anti-sense primer N5
Show；
4) to step 3) gained pcr amplification product purify, obtain the cDNA with the specific molecular label 5 ' end text
Storehouse.
6. the cDNA banking process that utilization transposase according to claim 5 is interrupted, it is characterised in that step 2) in, institute
State transposase embedding compound in annealing joint nucleotide sequence as shown in SEQ ID NO.6；Described transposase is Tn5
Family's transposase.
7. the cDNA banking process that the utilization transposase according to claim 5 or 6 is interrupted, it is characterised in that step 2) in,
Described cDNA fragmentations and plus jointing method include：Prepare reaction mixture：Transposase embedding is multiple described in claim 5 or 6
Compound 3-7 μ l, swivel base enzyme reaction buffer solution 4-8 μ l；Full-length cDNA is mixed with reaction mixture, is blown and beaten with pipettor and mixed；
The 50-58 DEG C of reaction 10-15min in PCR instrument.
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