CN103468670A - Full-length cDNA nucleic acid linear amplification method and kit - Google Patents
Full-length cDNA nucleic acid linear amplification method and kit Download PDFInfo
- Publication number
- CN103468670A CN103468670A CN2012101891659A CN201210189165A CN103468670A CN 103468670 A CN103468670 A CN 103468670A CN 2012101891659 A CN2012101891659 A CN 2012101891659A CN 201210189165 A CN201210189165 A CN 201210189165A CN 103468670 A CN103468670 A CN 103468670A
- Authority
- CN
- China
- Prior art keywords
- sequence
- nucleic acid
- cdna
- primer
- oligo
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the field of molecular biology, and particularly discloses a full-length cDNA nucleic acid linear amplification method. The method comprises the following steps: separating and purifying mRNA, carrying out reverse transcription synthesis of first strand cDNA, carrying out first strand cDNA 3' terminal extension to obtain full-length first strand cDNA, enriching and purifying the full-length strand cDNA, synthesizing double-stranded cDNA, and the like, such that the full-length cDNA with both known terminal sequences is synthesized. The full-length cDNA nucleic acid linear amplification method has characteristics of high sensitivity and simpleness, and is suitable for efficient synthesis of the full-length cDNA.
Description
Technical field
The present invention relates to biology field, be specifically related to a kind of full-length cDNA nucleic acid linear amplification method.
Background technology
The variation of gene expression dose and pattern is driving main biological procedures in organism.Separation and clone gene are the bases of research gene structure, function and expression.Set up in the past and screened that the classical way that carrys out the separating clone goal gene in cDNA and DNA library is loaded down with trivial details and workload is large.
The appearance of round pcr has improved the efficiency of separating clone goal gene greatly, the technology such as RT-PCR, panhandle PCR, ligation-mediated PCR provide shortcut for the unknown DNA fragmentation of amplification known array side, can be according to known array information design gene transformation primer, take the first chain cDNA of reverse transcription as template by PCR increase respectively 3 ' end and 5 ' terminal sequence clone, then splice full length sequence.Classical is rapid amplifying (rapid amplification of cDNA ends, the RACE) technology of cDNA end.Yet these method test cycles are normal, expense is high, and the problems such as pcr amplification efficiency is low often occur, and the chopped phenomenon of product often occurs, thereby utilize these methods to be not easy to obtain very much complete full-length gene, especially 5 ' of gene hold.
In addition, also there is the not enough problem of reversed transcriptive enzyme motility under low mRNA template amount in existing cDNA amplification technique, for example, when the RNA of denier (the mRNA template of fg level) is carried out to reverse transcription, reaction sensitivity is low, therefore, be badly in need of the reaction sensitivity that a kind of cDNA amplification method improves the cDNA amplification, obtain full-length cDNA.
Summary of the invention
The object of the invention is to overcome the defect of prior art, a pair of amplimer of the cDNA for the full-length cDNA that increases, a kind of highly sensitive, the simple cDNA nucleic acid of method linear amplification method and test kit are provided, efficient synthetic for full-length cDNA.
First aspect present invention discloses a kind of cDNA nucleic acid amplification method, the steps include:
(1) mRNA separation and purification;
(2) synthetic the first known chain cDNA of 5 ' terminal sequence of reverse transcription;
The first chain cDNA 3' end that (3) 5 ' terminal sequences are known extends, and obtains total length the first chain cDNA;
(4) total length the first chain cDNA enriching and purifying;
(5) the synthetic or RNA's of double-stranded cDNA transcribes.
Further, described step (1) is specially: get appropriate total RNA or cell pyrolysis liquid in reaction tubes, heat denatured, add biotin labeled Oligo dT joint primer after annealing; In reaction tubes, add the magnetic bead of affine Streptomycin sulphate mark to be reacted, collect the mRNA that magnetic bead obtains purifying.
Preferably, described denaturation temperature is 60～72 ℃, and the sex change time is 3～5min.Optimum, described denaturation temperature is 65 ℃.
Preferably, described annealing temperature is room temperature, and annealing time is 5～20min.Optimum, described annealing temperature is room temperature, annealing time is 8min.
Preferably, the temperature of reaction that the described magnetic bead that adds affine Streptomycin sulphate mark is reacted is room temperature, and the reaction times is 8～20min.Optimum, the reaction times is 10min.
Preferably, described collection magnetic bead is specially: after adding the magnetic bead of affine Streptomycin sulphate mark to be reacted, on the magnetic bead frame, collect magnetic bead, abandon supernatant, the magnetic bead of collecting with the buffered soln washing.
More excellent, collecting the magnetic bead time on described magnetic bead frame is 20～60s.Optimum, collecting the magnetic bead time on described magnetic bead frame is 30s.
More excellent, the SSC(sodium citrate buffer solution that described buffered soln is pH 7.2).
More excellent, described washing methods is to wash magnetic bead by the sodium citrate buffer of 0.5X and 0.1X successively.
Preferably, described biotin labeled Oligo dT joint primer is the Oligo dT joint primer that the 5' end is marked with vitamin H.Described Oligo dT joint primer length is 25～80bp, and its sequence comprises the left end joint sequence, oligomerization thymus pyrimidine sequence, and degenerate core nucleotide sequence VN successively from 5 ' end to 3 ' extreme direction.
Described joint (adaptor) sequence is the particular sequence be added on cDNA, is generally one section artificial synthesized sequence that sequence is known; Can the corresponding joints sequence synthesize the primer be complementary with its sequence, for the pcr amplification of cDNA.The described primer be complementary with joint sequence is called anchor primer.
More excellent, described Oligo dT joint primer length is 25～80bp, its sequence is: 5'-(N
1n
2na) T
1t
2t
dvN-3', wherein, N be tetra-kinds of thymus nucleic acid bases of ATCG any, a is that integer and span are 15～30; T is thymus pyrimidine, and d is that integer and span are 16～30; V be tri-kinds of thymus nucleic acid bases of ACG any; In bracket, sequence comprises the left end joint sequence, and the outer sequence of bracket is oligomerization thymus pyrimidine sequence and degenerate core nucleotide sequence VN.
Further, be provided with a plurality of restriction enzyme site sequences between described left end joint sequence and oligomerization thymus pyrimidine sequence.
Further, described Oligo dT joint primer length is 25～80bp, and its sequence is: 5'-(N
1n
2na) T
1t
2t
dvN-3': wherein, N be tetra-kinds of thymus nucleic acid bases of ATCG any, a is that integer and span are 15～30; T is thymus pyrimidine, and d is that integer and span are 16～30; V be tri-kinds of thymus nucleic acid bases of ACG any; In bracket, sequence comprises left end joint sequence and a plurality of restriction enzyme site sequence successively from 5 ' end to 3 ' extreme direction, and the outer sequence of bracket is oligomerization thymus pyrimidine sequence and degenerate core nucleotide sequence VN.
Further, the number of described restriction enzyme site is 1～6.
Further, described restriction enzyme site is selected from one or more in Sfi, EcoP151, BsgI or NotI.
Further, described Oligo dT joint primer starts to have 1～5 base to be modified from 5 ' end.Describedly be modified to that vitamin H coupling, LAN are modified and/or the fluorophor coupling is modified.
Optimum, described Oligo dT joint primer sequence is as shown in SEQ ID NO:3 or 6.
The described left end joint sequence of Oligo dT joint primer 5 ' end, be the known array of one section synthetic, can need designed, designed according to experiment.The effect of Oligo dT joint primer is the one section known array of 5 ' end connection at the first chain cDNA, obtains the first chain cDNA that 5 ' terminal sequence is known.Further, Oligo dT joint primer can also comprise a plurality of restriction enzyme site sequences, can insert restriction enzyme site at the end of cDNA, for the enzyme of cDNA cut, ligation transformed host cell.The biotin labeling of Oligo dT joint primer 5' end can pass through biotin-avidin Reaction Separation purified mRNA.
Preferably, described step (2) is specially: have to collection in the reaction tubes of magnetic bead and add the required reagent of reverse transcription to carry out reverse transcription reaction, after reaction finishes, degraded mRNA also collects magnetic bead, obtains the first known chain cDNA of 5 ' terminal sequence of purifying.
The required reagent of described reverse transcription is conventional, such as reverse transcription Buffer, reversed transcriptive enzyme, dNTP MIX, DTT etc.Preferably, this reversed transcriptive enzyme for sudden change after without the reversed transcriptive enzyme of RNase H activity the mixed enzyme with the archaeal dna polymerase with base mispairing identification and correct functioning.
More excellent, described step (2) reverse transcription reaction condition is 37～45 ℃, reacts 45～90 minutes.Optimum, described reverse transcription reaction condition is 37～45 ℃, reacts 90 minutes.
Step (2) degraded mRNA condition be routine, can be 37 ℃ of lower RNaseH enzymolysis 5～20 minutes.
More excellent, the reverse transcription reaction of described step (2) has also added cofactor.
Described cofactor is single stranded RNA, and length is 20～120bp, and its sequence is: 5 '-(N
1n
2n
b) A
1a
2a
e-3 '; Wherein, N be tetra-kinds of Yeast Nucleic Acid bases of AUCG any, b is that integer and span are 10～25; A is adenylic acid (AMP), and e is that integer and span are 12～25; In bracket, be the stochastic sequence of synthetic, and in bracket, the GC content of base sequence is that 40～60%, Tm value scope is 25～65 ℃, does not have the base of continuous 3 repetitions.
Further, the stochastic sequence in described bracket comprises 1～3 restriction enzyme site sequence.This restriction enzyme site can make that cofactor is cut at follow-up enzyme, remove in purification procedures, to after experiment do not exert an influence.
Further, the sequence of described cofactor is 5'-cgacguacguagcuagacuuaucguaaaaaaaaaaaaaaaaaa-3'(SEQ ID NO:1) or 5 '-gacgtacgtagctaggcctattcggccaaaaaaaaaaaaaaaaaa-3(SEQ ID NO:2).
Preferably, described step (3) is specially: add the Oligo Mdification primer in known the first chain cDNA of the 5 ' terminal sequence obtained to step (2), sex change, after annealing, add PCR to react required reagent and carry out cDNA 3' end extension, obtain total length the first chain cDNA that two terminal sequences are known.
It is conventional PCR reaction soln, for example MgCl that described PCR reacts required reagent
2, dNTP, PCR buffer, Taq polysaccharase and distilled water etc.
More excellent, described denaturation temperature is 68～75 ℃.Optimum, described denaturation temperature is 75 ℃.
More excellent, described annealing temperature is room temperature.
More excellent, described cDNA 3' end extension temperature is 16～80 ℃, the reaction times is 1～90min.
More excellent, described cDNA 3' end extension temperature is 25～40 ℃, the reaction times is 3～20min.Optimum, described cDNA 3' end extension temperature is 37 ℃, the reaction times is 10min.
Preferably, described Oligo Mdification primer length is 20～50bp, its sequence comprises right-hand member joint sequence and random primer sequence successively from 5 ' end to 3 ' extreme direction, and described Oligo Mdification primer 3 ' holds outermost base to be modified, or described Oligo Mdification primer 3 ' hold outermost base and with it continuous a plurality of bases modified.
More excellent, described Oligo Mdification primer length is 20～50bp, its sequence is: 5'-(N
1n
2nc) N
1n
2n
f-3'; Wherein, N be tetra-kinds of thymus nucleic acid bases of ATCG any, c is that integer and span are 18～30; F is that integer and span are 1～20; In bracket, sequence comprises the right-hand member joint sequence, the outer sequence of bracket is the random primer sequence, and described Oligo Mdification primer 3 ' holds outermost base to be modified, or described Oligo Mdification primer 3 ' hold outermost base and with it continuous a plurality of bases modified.
Further preferred, f is that integer and span are 6～12.
Further, also be provided with a plurality of restriction enzyme site sequences between described right-hand member joint sequence and random primer sequence.
Optimum, described Oligo Mdification primer length is 20～50bp, its sequence is: 5'-(N
1n
2nc) N
1n
2n
f-3'; Wherein, N be tetra-kinds of thymus nucleic acid bases of ATCG any, c is that integer and span are 18～30; F is that integer and span are 1～20; In bracket, sequence comprises right-hand member joint sequence and a plurality of restriction enzyme site sequence successively from 5 ' end to 3 ' extreme direction, the outer sequence of bracket is the random primer sequence, and described Oligo Mdification primer 3 ' holds outermost base to be modified, or described Oligo Mdification primer 3 ' hold outermost base and with it continuous a plurality of bases modified.
Further, the number of described restriction enzyme site is 1～6.
Further, described restriction enzyme site is selected from one or more in Sfi, EcoP151, BsgI or NotI.
Further, described modification is selected from amination, methylates, phosphorylation, two deoxidation, coupling vitamin H, LAN modifies and/or the coupling fluorophor is modified.
Optimum, described Oligo Mdification primer sequence is as shown in SEQ ID NO:4 or 7.
Oligo Mdification primer of the present invention, its 3 ' end is one section random primer sequence, can carry out random pair with the template in any source.Because Oligo Mdification primer 3 ' terminal bases is modified, therefore after the first chain cDNA annealed combination that this Oligo Mdification primer and 5 ' terminal sequence are known, only the cDNA chain be take the Oligo Mdification primer as the template continuation to 3 ' direction extension, and the chain at Oligo Mdification primer place does not increase.The right-hand member joint sequence of Oligo Mdification primer 5 ' end is the known array of one section synthetic, for obtaining the first chain cDNA that 3 ' terminal sequence is known.Further, because the Oligo Mdification primer also comprises a plurality of restriction enzyme site sequences, can insert restriction enzyme site at the other end of cDNA.
Right-hand member joint sequence in Oligo Mdification primer of the present invention both can be identical with the left end joint sequence in Oligo dT joint primer, also can be different.
Preferably, described step (4) is specially: the reaction solution heat denatured that previous step is obtained, collect total length the first chain cDNA that magnetic bead obtains purifying.
Described collection magnetic bead is specially: collect magnetic bead on the magnetic bead frame, abandon supernatant, the magnetic bead of collecting with the buffered soln washing.
More excellent, described step (4) denaturation temperature is 60～70 ℃.Optimum, described step (4) denaturation temperature is 65 ℃.
Preferably, described step (5) is specially: by method or the in-vitro transcription method of PCR, total length the first chain cDNA that the previous step of take obtains is template, design anchor primer, the synthetic known total length double-stranded DNA of two terminal sequences; The total length first chain cDNA of the purifying that the previous step of perhaps take obtains is template, the design anchor primer, and synthetic dsDNA, carry out the synthetic RNA of in-vitro transcription reaction after purifying.
The dsDNA(double-stranded DNA) synthetic can adopt low cycle P CR(Low Cycle PCR) method increased.Described low cycle P CR refer to cycle number lower PCR reaction, be generally less than 28 cycle numbers, do not reach the plateau of PCR, the cDNA product fidelity that low cycle P CR amplifies is good, the amount of cDNA is also increased.
The synthetic of RNA can, first by the synthetic dsDNA of the method for PCR, carry out the in-vitro transcription reaction, synthetic senseRNA, i.e. mRNA after purifying.
Second aspect present invention discloses a kind of cDNA nucleic acid amplification test kit, and described test kit comprises aforesaid Oligo Mdification primer and the Oligo dT joint primer be present in container.
More excellent, described test kit also comprises aforesaid cofactor.
Optimum, described test kit can also comprise various enzyme reagent, PCR buffered soln, one or more in dNTP, deionized water, RNase inhibitor.
Third aspect present invention, also disclose the application in eukaryote cDNA amplification of described cDNA nucleic acid amplification method and cDNA nucleic acid amplification test kit.
CDNA nucleic acid amplification method of the present invention and cDNA nucleic acid amplification test kit all can, for eukaryote, obtain the full-length cDNA that two terminal sequences are known.
The invention has the advantages that: 1), at Oligo dT reverse transcription primer 5' end mark vitamin H, in full-length cDNA nucleic acid linear amplification process, reaction product is carried out to enrichment, remove impurity; 2) utilize auxiliary RNA technology, solve the not enough defect of reversed transcriptive enzyme motility under low mRNA template amount, can carry out reverse transcription to the RNA of denier (being low to moderate the mRNA template of fg level), improved the sensitivity of cDNA building-up reactions; 3) by the mixed enzyme formula, reversed transcriptive enzyme without RNase H after sudden change is used with the archaeal dna polymerase combined optimization with base mispairing identification and correct functioning, the extension ability of reversed transcriptive enzyme is strengthened, coordinate cofactor, the mRNA template containing high GC is carried out to effective extension of long-chain; 4) for degraded or the incomplete mRNA of 5' end are slightly arranged, also can be extended; The final known full-length cDNA of two terminal sequences that obtains.
The accompanying drawing explanation
Fig. 1: full-length cDNA nucleic acid amplification method programchart
Fig. 2: PCR is electrophorogram (1.Marker 2. ordinary method amplified production 3-4. the inventive method amplified production 5. 6. U.S. E companies of U.S. C company) as a result
Fig. 3: PCR as a result electrophorogram (1.marker is followed successively by 5kb, 4kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp from top to bottom; 2-5. the method amplification of this experiment invention)
Fig. 4: PCR is electrophorogram (1. Chinese tallow tree seed specimen 2. aloe sample 3.Marker are followed successively by 5kb from top to bottom, 4kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp 4. clam sample 5. Penaeus vannamei sample 6. turbellarian worm samples) as a result
Embodiment
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is in order to describe specific specific embodiments, rather than in order to limit the scope of the invention; In specification sheets of the present invention and claims, unless explicitly pointed out in addition in literary composition, singulative " ", " one " and " this " comprise plural form.
When embodiment provides numerical range, unless should be understood that the present invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology of using in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.The concrete grammar used in embodiment, equipment, material, grasp and record of the present invention according to those skilled in the art to prior art, can also be with the method to described in the embodiment of the present invention, equipment, material is similar or any method, equipment and the material of the prior art that is equal to are realized the present invention.
Unless otherwise indicated, in the present invention, disclosed experimental technique, detection method, preparation method all adopt the routine techniques of molecular biology, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and the association area of the art routine.These technology are existing perfect explanation in existing document, specifically can be referring to MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; The series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromat in Protocol s (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
Further describe technical scheme of the present invention below by specific embodiment.
The synthetic of embodiment 1 micro-cDNA reacts with linear amplification
1. the preparation of total RNA
Gather the blade of paddy rice, put into the liquid nitrogen grinding powder, get the 100mg powder and use Trizol method (referring to the product manual explanation of Invitation company) to extract the total RNA of rice leaf.
2. using method
2.1 the inventive method
2.1.1 primer is synthetic
1) Oligo dT joint primer: 5 '-Biotin-GCCCAGCCAATCACCTAAAGTCAATTTTTTTTTTTTTTTTTTTVN-3'(SEQ ID NO:3).
2) cofactor: 5'-cgacguacguagcuagacuuaucguaaaaaaaaaaaaaaaaaa-3'(SEQ ID NO:1).
3) Oligo Mdification primer: 5'-GCCCAGCCAATCACCTAAAGTCANNNNNNNNN'-3 (SEQ ID NO:4), wherein NNNNNNNNN' is random primer, and Oligo Mdification primer 3 ' holds outermost base to be modified and process by amination.
4) anchor primer: 5'-GCCCAGCCAATCACCTAA-3'(SEQ ID NO:5)
2.1.2 full-length cDNA nucleic acid linear amplification method
(1) mRNA separation and purification: get two little centrifuge tubes, add respectively the total RNA of 2ng, carry out parallel control, 65 ℃ of sex change 5 minutes, add specially designed Oligo dT joint primer, add the SSC preparation method that SSC solution to final concentration is 0.5X(pH 7.2 to be: 87.7g NaCl and 44.1g Trisodium Citrate are dissolved in 500ml RNasefree water, dissolve and obtain), the mixing solutions room temperature is placed to annealing 8 minutes, magnetic bead (Streptavidine MagBead) 10 microlitres that add affine Streptomycin sulphate, room temperature reaction 10 minutes, on the magnetic bead frame, collect 30 seconds, abandon supernatant, successively with 0.5X and 0.1X SSC washing magnetic bead, collect the mRNA that magnetic bead obtains purifying, carry out next step experiment.
The known cDNA of (2) 5 ' terminal sequences is synthetic: have to collection in the reaction tubes of magnetic bead and add reverse transcription solution and cofactor, 90 minutes (the reverse transcription reaction system is in Table 1) of 42 ℃ of reactions, obtain mRNA-cDNA heterozygosis chain, after reverse transcription finishes, add 10U RNaseH reaction 10 minutes in reaction tubes, utilize enrichment with magnetic bead, remove supernatant, obtain cDNA.
Table 1 reverse transcription reaction system
The cDNA3' end that (3) 5 ' terminal sequences are known extends: in the cDNA obtained to previous step, add the Oligo Mdification primer, 75 ℃ of sex change, slowly be annealed to room temperature (room temperature is specially 20 ℃), add PCR reaction soln (reaction system is in Table 2) in reaction tubes, 37 ℃ of 10 minutes ends of the 3' to cDNA are extended, and obtain the first chain cDNA that two terminal sequences are known.
Table 23 ' end extension system
(4) total length the first chain cDNA enriching and purifying: by 65 ℃ of sex change of the purpose cDNA in reaction tubes, after sex change, reaction tubes is placed on the magnetic bead frame and collects magnetic bead, remove supernatant, total length the first chain cDNA after the acquisition purifying.
(5) the synthetic amplifying doulbe-chain cDNA of round pcr: add anchor primer, use high-fidelity DNA long fragment polysaccharase to carry out the PCR reaction, the PCR reaction system is in Table 3; The PCR reaction conditions is as follows: 95 ℃ of sex change in 5 minutes, 16 to 26 circulations, cycling condition is as follows: 95 ℃ 20 seconds, 65 ℃ 30 seconds, 72 ℃ 3 minutes.
Table 3PCR reaction system
2.2 simultaneous test
2.2.1 ordinary method: be the reverse transcription method of this area routine, specifically can be with reference to the method for RNA reverse transcription in " molecular cloning 3 ", amplification cDNA.
2.2.2SMART method: adopt Creator cDNA Library Construcion Kit test kit (purchased from U.S. Clontech company), by SMART method amplification cDNA.
2.2.3Mint method: adopt MINT-UniversalcDNA synthesis kit test kit (purchased from Russian Evrogene company), by Mint method amplification cDNA.2.3 experiment conclusion
The double-stranded cDNA that the double-stranded cDNA that the inventive method is obtained and method of contrast obtain carries out electrophoretic analysis, and experimental result is shown in Fig. 2.As shown in Figure 2, cDNA prepared by preparation method of the present invention compares with the amplified production of ordinary method, SMART method and Mint method, and the synthetic cDNA of present method is no matter from the cDNA total length or output all is better than other products.Described cDNA total length refers to that each gene pairs answers the length of the molecule of cDNA, and this is one of key factor determined RNA reverse transcription and the success or failure of cDNA amplification test, and generally, cDNA is longer represents that the integrity of gene is better.Therefore, known by the amplified production clip size that relatively each method obtains, the more existing ordinary method of full-length cDNA preparation method of the present invention, SMART method and Mint method are more excellent.
The synthetic of embodiment 2 micro-cDNA reacts with linear amplification
1. the preparation of total RNA
By gathering human placenta, put into the liquid nitrogen grinding powder, get 100mg and use Trizol method (referring to the product manual explanation of Invitation company) to extract human total rna.
2. method
2.1 primer is synthetic
1) Oligo dT joint primer: 5 '-Biotin-GCCCAGCCAATCACCTAAAGTCAA
ggccGAGGCggccAGCAGTgcagtTTTTTTTTTTTTTTTTTTVN-3'(SEQ ID NO:6), the restriction enzyme site sequence that wherein underscore is sfi, EcoP151 and BsgI.
2) cofactor: 5 '-gacguacguagcuaggccuauucggccaaaaaaaaaaaaaaaaaa-3'(SEQ ID NO:2)
3) Oligo Mdification primer: 5'-GCCCAGCCAATCACCTAAAGTCA
ggccgaggcggccnNNNNNN'N'N'-3(SEQ ID NO:7), wherein NNNNNNN'N'N' is random primer, and Oligo Mdification primer 3 ' holds outermost three continuous bases to be modified and process by two deoxidations; The restriction enzyme site sequence that underscore is sfi.
4) anchor primer: 5'-GCCCAGCCAATCACCTAA-3'(SEQ ID NO:5)
2.2 full-length cDNA nucleic acid linear amplification method
(1) separation and purification of mRNA: get 4 centrifuge tubes, every pipe is got the total RNA of 10pg, 60 ℃ of sex change 5 minutes, every Guan Jun adds Oligo dT joint primer, adding SSC solution to final concentration is 0.5X, the mixing solutions room temperature is placed to annealing 5 minutes, magnetic bead (Streptavidine MagBead) 10 microlitres that add affine Streptomycin sulphate, room temperature reaction 8 minutes, collect 20 seconds on the magnetic bead frame, abandon supernatant, respectively with 0.5X and 0.1X SSC washing magnetic bead, collect the mRNA that magnetic bead obtains purifying, carry out next step experiment.
Known the first chain cDNA of (2) 5 ' terminal sequences is synthetic: have to collection in 4 side reaction pipes of magnetic bead and add respectively reverse transcription solution and cofactor, 60 minutes (the reverse transcription reaction system is in Table 4) of 37 ℃ of reactions, obtain mRNA-cDNA heterozygosis chain, after reverse transcription finishes, add 10U RNaseH reaction 8 minutes in every side reaction pipe, utilize enrichment with magnetic bead, remove supernatant, obtain cDNA.
Table 4 reverse transcription reaction system
The known cDNA3' end of (3) 5 ' terminal sequences extends: add the Oligo Mdification primer in the known cDNA of the 5 ' terminal sequence obtained to previous step, 68 ℃ of sex change, slowly be annealed to room temperature; Add PCR reaction soln (reaction system is in Table 2) in reaction tubes, 37 ℃ of 10 minutes 3' ends to the first chain cDNA are extended, and obtain the purpose cDNA that two terminal sequences are known.
(4) total length the first chain cDNA enriching and purifying: by 70 ℃ of sex change of the purpose cDNA in reaction tubes, after sex change, reaction tubes is placed on the magnetic bead frame and collects magnetic bead, remove supernatant, total length the first chain cDNA after the acquisition purifying.
(5) the synthetic amplifying doulbe-chain cDNA of round pcr: add respectively the pcr amplification primer in 4 side reaction pipes, use high-fidelity DNA long fragment polysaccharase to carry out the PCR reaction, the PCR reaction system is in Table 3; The PCR reaction conditions is as follows: 95 ℃ of sex change in 5 minutes, 16 to 26 circulations, cycling condition is as follows: 95 ℃ 20 seconds, 65 ℃ 30 seconds, 72 ℃ 3 minutes.
2.3 experiment conclusion
The amplified production of getting in 4 arms carries out electrophoretic analysis, and electrophoresis result is shown in Fig. 3.Can the increase trace mrna template of animal-origin of the full-length cDNA nucleic acid linear amplification method of the present invention that shows placenta cDNA amplification, and the cDNA product total length of amplification experiment is fine with repeatability.
The synthetic of embodiment 3 micro-cDNA reacts with linear amplification
1. the preparation of total RNA
Gather turbellarian worm sample, Penaeus vannamei sample, clam sample, aloe sample, Chinese tallow tree seed specimen, use respectively the liquid nitrogen grinding powder, five kinds of samples are respectively got the 100mg powder and are used Trizol method (referring to the product manual explanation of Invitation company) to extract the total RNA of each sample.
2. experimental technique
2.1 primer is synthetic
1) Oligo dT joint primer: 5 '-Biotin-GCCCAGCCAATCACCTAAAGTCAA
ggccGAGGCggccAGCAGTgcagtTTTTTTTTTTTTTTTTTTVN-3'(SEQ ID NO:6), the restriction enzyme site sequence that wherein underscore is sfi, EcoP151 and BsgI.
2) cofactor: 5'-cgacguacguagcuagacuuaucguaaaaaaaaaaaaaaaaaa-3'(SEQ ID NO:1), the restriction enzyme site that wherein underscore is RsaI.
3) Oligo Mdification primer: 5'-GCCCAGCCAATCACCTAAAGTCANNNNNNNNN'-3(SEQ ID NO:4), wherein NNNNNNNNN' is random primer, and Oligo Mdification primer 3 ' holds outermost base to be modified and process by amination.
4) anchor primer: 5'-GCCCAGCCAATCACCTAA-3'(SEQ ID NO:5)
2.2 full-length cDNA nucleic acid linear amplification method
(1) mRNA separation and purification: get 5 little centrifuge tubes, the total RNA 10ng that adds a kind of sample in every centrifuge tube, 72 ℃ of sex change 3 minutes, add specially designed Oligo dT joint primer, adding SSC solution to final concentration is 0.5X, the mixing solutions room temperature is placed to annealing 8 minutes, magnetic bead (Streptavidine MagBead) 10 microlitres that add affine Streptomycin sulphate, room temperature reaction 20 minutes, on the magnetic bead frame, collect 25 seconds, abandon supernatant, respectively with 0.5X and 0.1X sodium citrate buffer solution washing magnetic bead, collect the mRNA that magnetic bead obtains purifying, carry out next step experiment.
Known the first chain cDNA of (2) 5 ' terminal sequences is synthetic: have to collection in 5 side reaction pipes of magnetic bead and add respectively reverse transcription solution and cofactor, 90 minutes (the reverse transcription reaction system is in Table 5) of 45 ℃ of reactions, obtain mRNA-cDNA heterozygosis chain, after reverse transcription finishes, add respectively 10U RNaseH reaction 5 minutes in reaction tubes, utilize enrichment with magnetic bead, remove supernatant, obtain the first chain cDNA that 5 ' terminal sequence is known.
Table 5 reverse transcription reaction system
The first chain cDNA3' end that (3) 5 ' terminal sequences are known extends: in the cDNA of the every kind of sample obtained to previous step, add respectively the Oligo Mdification primer, 75 ℃ of sex change, slowly be annealed to room temperature (room temperature is specially 20 ℃), add PCR reaction soln (reaction system is in Table 2) in reaction tubes, 37 ℃ of 10 minutes ends of the 3' to cDNA are extended, and obtain the first known chain cDNA of two terminal sequences of five kinds of samples.
(4) cDNA enriching and purifying: by 60 ℃ of sex change of the purpose cDNA in each reaction tubes, after sex change, reaction tubes is placed on the magnetic bead frame and collects magnetic bead, remove supernatant, the full-length cDNA of each sample after the acquisition purifying.
(5) the synthetic amplifying doulbe-chain cDNA of round pcr: add anchor primer in every centrifuge tube, use high-fidelity DNA long fragment polysaccharase to carry out the PCR reaction, the PCR reaction system is in Table 3; The PCR reaction conditions is as follows: 95 ℃ of sex change in 5 minutes, 16 to 26 circulations, cycling condition is as follows: 95 ℃ 20 seconds, 65 ℃ 30 seconds, 72 ℃ 3 minutes.
(6) the PCR product of five kinds of samples that previous step obtained is respectively got 3 μ l and is carried out electrophoresis, and electrophoresis result is shown in Fig. 4.
2.3 experiment conclusion
As shown in Figure 4, utilize amplification method of the present invention to carry out the cDNA amplification to the sample of different sources, the results show, method of the present invention is to plant Chinese tallow tree, aloe, marine organisms clam, prawn, and the different plant species such as the biological turbellarian worm in inland lake the RNA test amplification is preferably all arranged, and the cDNA total length that amplification obtains is better, cDNA output is also guaranteed, and the fidelity of linear amplification is good, is applicable to the eucaryon species of ocean, land.
Claims (17)
1. a cDNA nucleic acid amplification method, the steps include:
(1) mRNA separation and purification;
(2) synthetic the first known chain cDNA of 5 ' terminal sequence of reverse transcription;
The first chain cDNA 3' end that (3) 5 ' terminal sequences are known extends, and obtains total length the first chain cDNA;
(4) total length the first chain cDNA enriching and purifying;
(5) the synthetic or RNA's of double-stranded cDNA transcribes.
2. nucleic acid amplification method as claimed in claim 1, is characterized in that, described step (1) is specially: get appropriate total RNA or cell pyrolysis liquid in reaction tubes, heat denatured, add biotin labeled Oligo dT joint primer after annealing; In reaction tubes, add the magnetic bead of affine Streptomycin sulphate mark to be reacted, collect the mRNA that magnetic bead obtains purifying.
3. nucleic acid amplification method as claimed in claim 2, it is characterized in that, described Oligo dT joint primer length is 25～80bp, and its sequence comprises the left end joint sequence successively from 5 ' end to 3 ' extreme direction, oligomerization thymus pyrimidine sequence, and degenerate core nucleotide sequence VN.
4. nucleic acid amplification method as claimed in claim 2, is characterized in that, described Oligo dT joint primer length is 25～80bp, and its sequence is: 5'-(N
1n
2na) T
1t
2t
dvN-3', wherein, N be tetra-kinds of thymus nucleic acid bases of ATCG any, a is that integer and span are 15～30; T is thymus pyrimidine, and d is that integer and span are 16～30; V be tri-kinds of thymus nucleic acid bases of ACG any; In bracket, sequence comprises the left end joint sequence, and the outer sequence of bracket is oligomerization thymus pyrimidine sequence and degenerate core nucleotide sequence VN.
5. the described nucleic acid amplification method of claim as arbitrary as claim 3 or 4, is characterized in that, between described left end joint sequence and oligomerization thymus pyrimidine sequence, is provided with a plurality of restriction enzyme site sequences.
6. nucleic acid amplification method as claimed in claim 1, it is characterized in that, described step (2) is specially: have to collection in the reaction tubes of magnetic bead and add the required reagent of reverse transcription to carry out reverse transcription reaction, reaction finishes rear degraded mRNA and collects magnetic bead, obtains the first known chain cDNA of 5 ' terminal sequence of purifying.
7. the nucleic acid amplification method of stating as claim 6, is characterized in that, the reverse transcription reaction of described step (2) has also added cofactor, and described cofactor is single stranded RNA, and length is 20～120bp, and its sequence is: 5 '-(N
1n
2n
b) A
1a
2a
e-3 '; Wherein, N be tetra-kinds of Yeast Nucleic Acid bases of AUCG any, b is that integer and span are 10～25; A is adenylic acid (AMP), and e is that integer and span are 12～25; In bracket, be the stochastic sequence of synthetic, and in bracket, the GC content of base sequence is that 40～60%, Tm value scope is 25～65 ℃, does not have the base of continuous 3 repetitions.
8. nucleic acid amplification method as claimed in claim 7, is characterized in that, described cofactor sequence is as shown in SEQ ID NO:1 or 2.
9. nucleic acid amplification method as claimed in claim 1, it is characterized in that, described step (3) is specially: in known the first chain cDNA of the 5 ' terminal sequence obtained to step (2), add the Oligo Mdification primer, sex change, after annealing, add PCR to react required reagent and carry out cDNA 3' end extension, obtain total length the first chain cDNA that two terminal sequences are known.
10. nucleic acid amplification method as claimed in claim 9, it is characterized in that, described Oligo Mdification primer length is 20～50bp, its sequence comprises right-hand member joint sequence and random primer sequence successively from 5 ' end to 3 ' extreme direction, and described Oligo Mdification primer 3 ' holds outermost base to be modified, or described Oligo Mdification primer 3 ' hold outermost base and with it continuous a plurality of bases modified.
11. nucleic acid amplification method as claimed in claim 9, is characterized in that, described Oligo Mdification primer length is 20～50bp, and its sequence is: 5'-(N
1n
2nc) N
1n
2n
f-3'; Wherein, N be tetra-kinds of thymus nucleic acid bases of ATCG any, c is that integer and span are 18～30; F is that integer and span are 1～20; In bracket, sequence comprises the right-hand member joint sequence, the outer sequence of bracket is the random primer sequence, and described Oligo Mdification primer 3 ' holds outermost base to be modified, or described Oligo Mdification primer 3 ' hold outermost base and with it continuous a plurality of bases modified.
12. the described nucleic acid amplification method of claim as arbitrary as claim 10 or 11, is characterized in that, also is provided with a plurality of restriction enzyme site sequences between described right-hand member joint sequence and random primer sequence.
13. nucleic acid amplification method as claimed in claim 1, is characterized in that, described step (4) is specially: the reaction solution heat denatured that previous step is obtained, collect total length the first chain cDNA that magnetic bead obtains purifying.
14. nucleic acid amplification method as claimed in claim 1, it is characterized in that, described step (5) is specially: by method or the in-vitro transcription method of PCR, total length the first chain cDNA that the previous step of take obtains is template, the design anchor primer, the synthetic known total length double-stranded DNA of two terminal sequences; The total length first chain cDNA of the purifying that the previous step of perhaps take obtains is template, the design anchor primer, and synthetic dsDNA, carry out the synthetic RNA of in-vitro transcription reaction after purifying.
A 15. cDNA nucleic acid amplification test kit, it is characterized in that, described test kit comprises the Oligo Mdification primer described in the Oligo dT joint primer described in the arbitrary claim of the claim 3-5 be present in container and the arbitrary claim of claim 10-12.
16. test kit, is characterized in that as claimed in claim 15, also comprises the cofactor described in the arbitrary claim of claim 7 or 8 in described test kit.
17. the application of test kit in eukaryote cDNA amplification as described in amplification method or the arbitrary claim of claim 15-16 as described in claim as arbitrary as claim 1-14.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210189165.9A CN103468670B (en) | 2012-06-08 | 2012-06-08 | Full-length cDNA nucleic acid linear amplification method and test kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210189165.9A CN103468670B (en) | 2012-06-08 | 2012-06-08 | Full-length cDNA nucleic acid linear amplification method and test kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103468670A true CN103468670A (en) | 2013-12-25 |
| CN103468670B CN103468670B (en) | 2016-01-20 |
Family
ID=49793711
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210189165.9A Active CN103468670B (en) | 2012-06-08 | 2012-06-08 | Full-length cDNA nucleic acid linear amplification method and test kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103468670B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087765A (en) * | 2014-05-16 | 2015-11-25 | 深圳先进技术研究院 | Polythymine template, fluorescent copper nano-cluster based on same, preparation method of fluorescent copper nano-cluster and ATP detection method |
| CN106554959A (en) * | 2017-01-17 | 2017-04-05 | 北京全式金生物技术有限公司 | A kind of reverse transcription primer suitable for efficient reverse transcription reaction is combined |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001059101A1 (en) * | 2000-02-10 | 2001-08-16 | The Penn State Research Foundation | Method for amplifying full length single strand polynucleotide sequences |
| CN101864413A (en) * | 2010-05-27 | 2010-10-20 | 东北农业大学 | 5'-RACE joint sequence addition method as well as joint sequence and amplification method of 5' end unknown gene complete encoding sequence |
-
2012
- 2012-06-08 CN CN201210189165.9A patent/CN103468670B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001059101A1 (en) * | 2000-02-10 | 2001-08-16 | The Penn State Research Foundation | Method for amplifying full length single strand polynucleotide sequences |
| CN101864413A (en) * | 2010-05-27 | 2010-10-20 | 东北农业大学 | 5'-RACE joint sequence addition method as well as joint sequence and amplification method of 5' end unknown gene complete encoding sequence |
Non-Patent Citations (3)
| Title |
|---|
| ELIZABETH SCOTTO–LAVINO ET AL.: "5" end cDNA amplification using classic RACE", 《NATURE PROTOCOLS》, vol. 1, no. 6, 29 December 2006 (2006-12-29), pages 2555 - 2562, XP009153012 * |
| 段静波 等: "cDNA末端快速扩增试剂盒研发进展", 《生命的化学》, vol. 28, no. 5, 31 December 2008 (2008-12-31), pages 641 - 647 * |
| 罗聪 等: "一种高效获取基因5"末端的RACE方法", 《植物生理学报》, vol. 47, no. 4, 31 December 2011 (2011-12-31), pages 409 - 414 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087765A (en) * | 2014-05-16 | 2015-11-25 | 深圳先进技术研究院 | Polythymine template, fluorescent copper nano-cluster based on same, preparation method of fluorescent copper nano-cluster and ATP detection method |
| CN106554959A (en) * | 2017-01-17 | 2017-04-05 | 北京全式金生物技术有限公司 | A kind of reverse transcription primer suitable for efficient reverse transcription reaction is combined |
| CN106554959B (en) * | 2017-01-17 | 2019-08-13 | 北京全式金生物技术有限公司 | A kind of reverse transcription primer combination suitable for efficient reverse transcription reaction |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103468670B (en) | 2016-01-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200232030A1 (en) | Massively multiplexed rna sequencing | |
| Zhang et al. | Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly (A) selection | |
| RU2565550C2 (en) | Direct capture, amplification and sequencing of target dna using immobilised primers | |
| CN102124126A (en) | Cdna synthesis using non-random primers | |
| US20080131937A1 (en) | Conversion of Target Specific Amplification to Universal Sequencing | |
| JP7033602B2 (en) | Barcoded DNA for long range sequencing | |
| EP3650559B1 (en) | Direct rna nanopore sequencing with help of a stem-loop polynucleotide | |
| CN107201408B (en) | Method for developing sisal hemp SSR primer based on transcriptome sequencing | |
| CN108611398A (en) | Genotyping is carried out by new-generation sequencing | |
| EA005577B1 (en) | A material having an immobilized nucleic acid prepared by the method using a chimeric oligonucleotide primer, a dna polymerase and an endonuclease | |
| US11761037B1 (en) | Probe and method of enriching target region applicable to high-throughput sequencing using the same | |
| EP3289105B1 (en) | Compositions and methods for constructing strand specific cdna libraries | |
| US20090023151A1 (en) | Method For The Labeling And Detection Of Small Polynucleotides | |
| US11820980B2 (en) | Methods and compositions for preparing nucleic acid sequencing libraries | |
| JP2024099616A (en) | Sequence determination method for genome rearrangement detection | |
| CN104630211B (en) | A kind of construction method of Small RNA cDNA libraries | |
| WO2007142608A1 (en) | Nucleic acid concatenation | |
| DE69736475D1 (en) | QUANTIFYING RNA TRANSCRIPTS USING GENOMIC DNA AS THE INTERNAL STANDARD OF THE AMPLIFICATION REACTION | |
| CN103468670B (en) | Full-length cDNA nucleic acid linear amplification method and test kit | |
| CA2073184A1 (en) | Compositions and methods for analyzing genomic variation | |
| CN116287124A (en) | Single-stranded joint pre-connection method, library construction method of high-throughput sequencing library and kit | |
| US20240247302A1 (en) | Massive generation of chemically ligateable probes for multiplexed fish | |
| KR20150090352A (en) | Method of collecting nucleic acid fragments separated from the sequencing process | |
| Mondal et al. | High throughput DNA sequencing and its implication in plant science research | |
| CN106676096A (en) | Whole set of reagents and method for cloning full-length gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |