CN103468670B - Full-length cDNA nucleic acid linear amplification method and test kit - Google Patents
Full-length cDNA nucleic acid linear amplification method and test kit Download PDFInfo
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Abstract
The present invention relates to biology field, specifically disclose a kind of full-length cDNA nucleic acid linear amplification method, by mRNA separation and purification; Reverse transcription synthesizes the first chain cDNA; First chain cDNA? 3 ˊ ends extend, and obtain total length first chain cDNA; Total length first chain cDNA enriching and purifying; The steps such as the synthesis of double-strand cDNA, synthesize the full-length cDNA that two terminal sequences are known.Full-length cDNA nucleic acid linear amplification method of the present invention is highly sensitive, method simple, is applicable to the efficient synthesis of 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 change of gene expression dose and pattern drives biological procedures main in organism.To be separated and clone gene is the basis studying gene structure, function and expression.Set up in the past and screened cDNA and DNA library and came that the classical way of separating clone goal gene is loaded down with trivial details and workload is large.
The appearance of round pcr greatly improves the efficiency of separating clone goal gene, the technology such as RT-PCR, panhandle PCR, ligation-mediated PCR are that the unknown DNA fragmentation of amplification known array side provides shortcut, can according to known array information design gene transformation primer, to be increased respectively 3 ' end and 5 ' terminal sequence cloning by PCR for template with the first chain cDNA of reverse transcription, then splice full length sequence.Rapid amplifying (rapidamplificationofcDNAends, the RACE) technology of classical is cDNA end.But these method test cycles often, costly, and often there is the problems such as pcr amplification efficiency is low, and often occur the short phenomenon of cutting of product, thus utilize these methods to be not easy to obtain very much complete full-length gene, especially 5 ' end of gene.
In addition, also there is the problem that reversed transcriptive enzyme motility is not enough under low mRNA template amount in existing cDNA amplification technique, when carrying out reverse transcription to the RNA of denier (the mRNA template of such as fg level), reaction sensitivity is low, therefore, be badly in need of a kind of cDNA amplification method to improve the reaction sensitivity of cDNA amplification, obtain full-length cDNA.
Summary of the invention
The object of the invention is to the defect overcoming prior art, provide a pair cDNA amplimer for the full-length cDNA that increases, a kind of highly sensitive, method simple cDNA nucleic acid linear amplification method and test kit, for the efficient synthesis of 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) the first chain cDNA that reverse transcription synthesis 5 ' terminal sequence is known;
The first chain cDNA3' end that (3) 5 ' terminal sequences are known extends, and obtains total length first chain cDNA;
(4) total length first chain cDNA enriching and purifying;
(5) synthesis of double-strand cDNA or transcribing of RNA.
Further, described step (1) is specially: get appropriate total serum IgE or cell pyrolysis liquid in reaction tubes, heat denatured, adds biotin labeled OligodT adapter-primer after annealing; The magnetic bead adding affine Streptomycin sulphate mark in reaction tubes reacts, and collects the mRNA that magnetic bead obtains purifying.
Preferably, described denaturation temperature is 60 ~ 72 DEG C, and denaturation time is 3 ~ 5min.Optimum, described denaturation temperature is 65 DEG C.
Preferably, described annealing temperature is room temperature, and annealing time is 5 ~ 20min.Optimum, described annealing temperature is room temperature, and annealing time is 8min.
Preferably, described in add affine Streptomycin sulphate mark the magnetic bead temperature of reaction of carrying out reacting be room temperature, the reaction times is 8 ~ 20min.Optimum, the reaction times is 10min.
Preferably, described collection magnetic bead is specially: add affine Streptomycin sulphate mark magnetic bead react after, on magnetic bead frame, collect magnetic bead, abandon supernatant, wash the magnetic bead collected with buffered soln.
More excellent, described magnetic bead frame being collected the magnetic bead time is 20 ~ 60s.Optimum, described magnetic bead frame being collected the magnetic bead time is 30s.
More excellent, described buffered soln is the SSC(sodium citrate buffer solution of pH7.2).
More excellent, described washing methods is, successively with the sodium citrate buffer washing magnetic bead of 0.5X and 0.1X.
Preferably, described biotin labeled OligodT adapter-primer is the OligodT adapter-primer that 5' end is marked with vitamin H.Described OligodT adapter-primer length is 25 ~ 80bp, and its sequence comprises left end joint sequence, oligomerization thymus pyrimidine sequence successively from 5 ' end to 3 ' extreme direction, and degenerate core nucleotide sequence VN.
Described joint (adaptor) sequence is the particular sequence be added on cDNA, is generally the artificial synthesized sequence that one section of sequence is known; Can the primer that matches of corresponding joints sequent synthesis and its sequence, for the pcr amplification of cDNA.The described primer matched with joint sequence is called anchor primer.
More excellent, described OligodT adapter-primer length is 25 ~ 80bp, and its sequence is: 5'-(N
1n
2na) T
1t
2t
dvN-3', wherein, N be ATCG tetra-kinds of thymus nucleic acid bases any one, a is integer and span is 15 ~ 30; T is thymus pyrimidine, and d is integer and span is 16 ~ 30; V be ACG tri-kinds of thymus nucleic acid bases any one; In bracket, sequence comprises left end joint sequence, and the outer sequence of bracket is oligomerization thymus pyrimidine sequence and degenerate core nucleotide sequence VN.
Further, multiple restriction enzyme site sequence is provided with between described left end joint sequence and oligomerization thymus pyrimidine sequence.
Further, described OligodT adapter-primer length is 25 ~ 80bp, and its sequence is: 5'-(N
1n
2na) T
1t
2t
dvN-3': wherein, N be ATCG tetra-kinds of thymus nucleic acid bases any one, a is integer and span is 15 ~ 30; T is thymus pyrimidine, and d is integer and span is 16 ~ 30; V be ACG tri-kinds of thymus nucleic acid bases any one; In bracket, sequence comprises left end joint sequence and multiple 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 OligodT adapter-primer starts have 1 ~ 5 base to be modified from 5 ' end.Described be modified to vitamin H coupling, LAN modify and/or fluorophor coupling modify.
Optimum, described OligodT adapter-primer sequence is as shown in SEQIDNO:3 or 6.
The described left end joint sequence that OligodT adapter-primer 5 ' is held, is the known array of one section of synthetic, experimentally can needs designed, designed.The effect of OligodT adapter-primer is 5 ' end connection, the one section of known array at the first chain cDNA, obtains the first chain cDNA that 5 ' terminal sequence is known.Further, OligodT adapter-primer can also comprise multiple restriction enzyme site sequence, and can insert restriction enzyme site in one end of cDNA, the enzyme for cDNA is cut, ligation transformed host cell.The biotin labeling that OligodT adapter-primer 5' holds is by biotin-avidin Reaction Separation purified mRNA.
Preferably, described step (2) is specially: have to collection in the reaction tubes of magnetic bead and add reagent needed for reverse transcription and carry out reverse transcription reaction, and reaction terminates rear degraded mRNA and collects magnetic bead, the first chain cDNA that 5 ' terminal sequence of acquisition purifying is known.
Reagent needed for described reverse transcription is conventional, such as reverse transcription Buffer, reversed transcriptive enzyme, dNTPMIX, DTT etc.Preferably, this reversed transcriptive enzyme is the mixed enzyme of the rear reversed transcriptive enzyme without RNaseH activity of sudden change and the archaeal dna polymerase with base mispairing identification and correct functioning.
More excellent, described step (2) reverse transcription reaction condition is 37 ~ 45 DEG C, reacts 45 ~ 90 minutes.Optimum, described reverse transcription reaction condition is 37 ~ 45 DEG C, reacts 90 minutes.
Step (2) degraded mRNA condition be routine, can at 37 DEG C RNaseH enzymolysis 5 ~ 20 minutes.
More excellent, the reverse transcription reaction of described step (2) also adds 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 AUCG tetra-kinds of ribonucleic acid base any one, b is integer and span is 10 ~ 25; A is adenylic acid (AMP), and e is integer and span is 12 ~ 25; Be the stochastic sequence of synthetic in bracket, and in bracket, the GC content of base sequence is 40 ~ 60%, Tm value scope is 25 ~ 65 DEG C, there are not the bases 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, does not have an impact to experiment afterwards.
Further, the sequence of described cofactor is 5'-cgacguacguagcuagacuuaucguaaaaaaaaaaaaaaaaaa-3'(SEQIDN O:1) or 5 '-gacgtacgtagctaggcctattcggccaaaaaaaaaaaaaaaaaa-3(SEQIDNO: 2).
Preferably, described step (3) is specially: add Oligo Mdification primer, sex change, after annealing in the first chain cDNA that the 5 ' terminal sequence obtained to step (2) is known, add the required reagent of PCR reaction and carry out cDNA3' end extension, obtain two terminal sequences known total length first chain cDNA.
The required reagent of described PCR reaction is Standard PCR reaction soln, such as MgCl
2, dNTP, PCRbuffer, Taq polysaccharase and distilled water etc.
More excellent, described denaturation temperature is 68 ~ 75 DEG C.Optimum, described denaturation temperature is 75 DEG C.
More excellent, described annealing temperature is room temperature.
More excellent, described cDNA3' end extension temperature is 16 ~ 80 DEG C, and the reaction times is 1 ~ 90min.
More excellent, described cDNA3' end extension temperature is 25 ~ 40 DEG C, and the reaction times is 3 ~ 20min.Optimum, described cDNA3' end extension temperature is 37 DEG C, and 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 the multiple base of continuous print modified.
More excellent, described Oligo Mdification primer length is 20 ~ 50bp, and its sequence is: 5'-(N
1n
2nc) N
1n
2n
f-3'; Wherein, N be ATCG tetra-kinds of thymus nucleic acid bases any one, c is integer and span is 18 ~ 30; F is integer and span is 1 ~ 20; In bracket, sequence comprises right-hand member joint sequence, the outer sequence of bracket is 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 the multiple base of continuous print modified.
Preferred further, f is integer and span is 6 ~ 12.
Further, multiple restriction enzyme site sequence is also provided with between described right-hand member joint sequence and random primer sequence.
Optimum, described Oligo Mdification primer length is 20 ~ 50bp, and its sequence is: 5'-(N
1n
2nc) N
1n
2n
f-3'; Wherein, N be ATCG tetra-kinds of thymus nucleic acid bases any one, c is integer and span is 18 ~ 30; F is integer and span is 1 ~ 20; In bracket, sequence comprises right-hand member joint sequence and multiple restriction enzyme site sequence successively from 5 ' end to 3 ' extreme direction, the outer sequence of bracket is 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 the multiple base of continuous print 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, couple biotin, LAN modifies and/or coupling fluorophor is modified.
Optimum, described Oligo Mdification primer sequence is as shown in SEQIDNO:4 or 7.
Oligo Mdification primer of the present invention, its 3 ' end is one section of random primer sequence, can carry out random pair with the template of originating arbitrarily.Because Oligo Mdification primer 3 ' terminal bases is modified, therefore after this Oligo Mdification primer and known the first chain cDNA annealed combination of 5 ' terminal sequence, only cDNA chain is with Oligo Mdification primer for template continues to extend to 3 ' direction, and the chain at Oligo Mdification primer place does not increase.The right-hand member joint sequence that Oligo Mdification primer 5 ' is held is the known array of one section of synthetic, for obtaining the first known chain cDNA of 3 ' terminal sequence.Further, because Oligo Mdification primer also comprises multiple restriction enzyme site sequence, restriction enzyme site can be inserted 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 OligodT adapter-primer, also can be different.
Preferably, described step (4) is specially: the reaction solution heat denatured obtained previous step, collects the total length first chain cDNA that magnetic bead obtains purifying.
Described collection magnetic bead is specially: on magnetic bead frame, collect magnetic bead, abandon supernatant, washs the magnetic bead collected with buffered soln.
More excellent, described step (4) denaturation temperature is 60 ~ 70 DEG C.Optimum, described step (4) denaturation temperature is 65 DEG C.
Preferably, described step (5) is specially: by method or the in-vitro transcription method of PCR, and the total length first chain cDNA obtained with previous step is for template, and design anchor primer, synthesizes the total length double-stranded DNA that two terminal sequences are known; Or the total length first chain cDNA of the purifying obtained with previous step is for template, design anchor primer, synthesis dsDNA, carries out in-vitro transcription Reactive Synthesis RNA after purifying.
DsDNA(double-stranded DNA) synthesis can adopt low cycle P CR(LowCyclePCR) method increase.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, and the amount of cDNA is also increased.
The synthesis of RNA first by the method synthesis dsDNA of PCR, can carry out in-vitro transcription reaction after purifying, synthesis senseRNA, i.e. mRNA.
Second aspect present invention discloses a kind of cDNA nucleic acid amplification kit, and described test kit comprises and is present in aforesaid Oligo Mdification primer in container and OligodT adapter-primer.
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, RNaseinhibitor.
Third aspect present invention, also discloses described cDNA nucleic acid amplification method and the application of cDNA nucleic acid amplification kit in eukaryote cDNA increases.
CDNA nucleic acid amplification method of the present invention and cDNA nucleic acid amplification kit all for eukaryote, can obtain the full-length cDNA that two terminal sequences are known.
The invention has the advantages that: 1) at OligodT reverse transcription primer 5' end mark vitamin H, in full-length cDNA nucleic acid linear amplification process, enrichment is carried out to reaction product, removing impurity; 2) utilize auxiliary RNA technology, solve the defect of reversed transcriptive enzyme motility deficiency under low mRNA template amount, reverse transcription can be carried out to the RNA of denier (being low to moderate the mRNA template of fg level), improve the sensitivity of cDNA building-up reactions; 3) filled a prescription by mixed enzyme, use after sudden change without the reversed transcriptive enzyme of RNaseH and the archaeal dna polymerase combined optimization with base mispairing identification and correct functioning, the extension ability of reversed transcriptive enzyme is strengthened, coordinates cofactor, the mRNA template containing high GC is carried out to effective extension of long-chain; 4) for slightly degrading or the incomplete mRNA of 5' end, also can extend; The full-length cDNA that final acquisition two terminal sequence is known.
Accompanying drawing explanation
Fig. 1: full-length cDNA nucleic acid amplification method programchart
Fig. 2: PCR result electrophorogram (E company of the U.S. of C company 6. of 1.Marker2. ordinary method amplified production 3-4. the inventive method amplified production 5. U.S.)
(1.marker is followed successively by 5kb to Fig. 3: PCR result electrophorogram from top to bottom, 4kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp; The method amplification of this experiment of 2-5. invention)
Fig. 4: PCR result electrophorogram (1. Chinese tallow tree seed specimen 2. aloe sample 3.Marker is followed successively by 5kb from top to bottom, 4kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp4. clam sample 5. Penaeus vannamei sample 6. turbellarian worm sample)
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 to describe specific specific embodiments, instead of 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, should be understood that except non-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 used in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and association area.These technology are existing in existing document improves explanation, specifically can see the MOLECULARCLONING:ALABORATORYMANUAL such as Sambrook, Secondedition, ColdSpringHarborLaboratoryPress, 1989andThirdedition, 2001; Ausubel etc., CURRENTPROTOCOLSINMOLECULARBIOLOGY, JohnWiley & Sons, NewYork, 1987andperiodicupdates; TheseriesMETHODSINENZYMOLOGY, AcademicPress, SanDiego; Wolffe, CHROMATINSTRUCTUREANDFUNCTION, Thirdedition, AcademicPress, SanDiego, 1998; METHODSINENZYMOLOGY, Vol.304, Chromatin (P.M.WassarmanandA.P.Wolffe, eds.), AcademicPress, SanDiego, 1999; And METHODSINMOLECULARBIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) HumanaPress, Totowa, 1999 etc.
Technical scheme of the present invention is further described below by specific embodiment.
Synthesis and the linear amplification of the micro-cDNA of embodiment 1 react
1. the preparation of total serum IgE
Gather the blade of paddy rice, put into liquid nitrogen grinding powder, get 100mg powder and use Trizol method (illustrating see Invitation Products handbook) to extract rice leaf total serum IgE.
2. using method
2.1 the inventive method
2.1.1 primer synthesis
1) OligodT adapter-primer: 5 '-Biotin-GCCCAGCCAATCACCTAAAGTCAATTTTTTTTTTTTTTTTTTTVN-3'(SEQIDNO:3).
2) cofactor: 5'-cgacguacguagcuagacuuaucguaaaaaaaaaaaaaaaaaa-3'(SEQIDN O:1).
3) Oligo Mdification primer: 5'-GCCCAGCCAATCACCTAAAGTCANNNNNNNNN'-3 (SEQIDNO:4), wherein NNNNNNNNN' is random primer, and Oligo Mdification primer 3 ' holds outermost base by amination moditied processing.
4) anchor primer: 5'-GCCCAGCCAATCACCTAA-3'(SEQIDNO:5)
2.1.2 full-length cDNA nucleic acid linear amplification method
(1) mRNA separation and purification: get two little centrifuge tubes, add 2ng total serum IgE respectively, carry out parallel control, 65 DEG C of sex change 5 minutes, add specially designed OligodT adapter-primer, to add SSC solution to final concentration be the SSC preparation method of 0.5X(pH7.2 is: 87.7gNaCl and 44.1g Trisodium Citrate is dissolved in 500mlRNasefree water, dissolve and obtain), mixing solutions room temperature is placed annealing 8 minutes, add magnetic bead (StreptavidineMagBead) 10 microlitre of affine Streptomycin sulphate, room temperature reaction 10 minutes, collect 30 seconds on magnetic bead frame, abandon supernatant, magnetic bead is washed successively with 0.5X and 0.1XSSC, collect the mRNA that magnetic bead obtains purifying, carry out next step experiment.
The cDNA synthesis that (2) 5 ' terminal sequences are known: have to collection in the reaction tubes of magnetic bead and add reverse transcription solution and cofactor, 42 DEG C of reactions, 90 minutes (reverse transcription reaction system is in table 1), obtain mRNA-cDNA heterozygosis chain, after reverse transcription terminates, in reaction tubes, add 10URNaseH react 10 minutes, 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 that previous step obtains, add Oligo Mdification primer, 75 DEG C of sex change, slowly be annealed to room temperature (room temperature is specially 20 DEG C), PCR reaction soln (reaction system is in table 2) is added in reaction tubes, 37 DEG C extend the 3' end of cDNA for 10 minutes, obtain the first chain cDNA that two terminal sequences are known.
Table 23 ' end extension system
(4) total length first chain cDNA enriching and purifying: by DEG C sex change of the object cDNA65 in reaction tubes, is placed in reaction tubes on magnetic bead frame after sex change and collects magnetic bead, remove supernatant, obtains the total length first chain cDNA after purifying.
(5) round pcr synthesis amplifying doulbe-chain cDNA: add anchor primer, use high-fidelity DNA long fragment polysaccharase to carry out PCR reaction, PCR reaction system is in table 3; PCR reaction conditions is as follows: 95 DEG C of sex change in 5 minutes, 16 to 26 circulations, and cycling condition is as follows: 95 DEG C 20 seconds, 65 DEG C 30 seconds, 72 DEG C 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 with reference to the method for RNA reverse transcription in " molecular cloning 3 ", amplification cDNA.
2.2.2SMART method: adopt CreatorcDNALibraryConstrucionKit test kit (purchased from American Clontech company), by SMART method amplification cDNA.
2.2.3Mint method: adopt MINT-UniversalcDNAsynthesiskit test kit (purchased from Russian Evrogene company), by Mint method amplification cDNA.2.3 experiment conclusion
The double-strand cDNA that the double-strand cDNA obtain the inventive method 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 is compared with the amplified production of ordinary method, SMART method and Mint method, and no matter the cDNA of present method synthesis is all better than other products from cDNA total length or output.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 determining RNA reverse transcription and the success or failure of cDNA amplification test, and generally, cDNA is longer, and to represent the integrity of gene better.Therefore, the amplified production clip size obtained by more each method is known, and the more existing ordinary method of full-length cDNA preparation method of the present invention, SMART method and Mint method are more excellent.
Synthesis and the linear amplification of the micro-cDNA of embodiment 2 react
1. the preparation of total serum IgE
By gathering human placenta, putting into liquid nitrogen grinding powder, getting 100mg and using Trizol method (illustrating see Invitation Products handbook) to extract human total rna.
2. method
2.1 primer synthesis
1) OligodT adapter-primer: 5 '-Biotin-GCCCAGCCAATCACCTAAAGTCAA
ggccGAGGCggccAGCAGTgcagtTTTTTTTTTTTTTTTTTTVN-3'(SEQIDNO:6), wherein underscore is the restriction enzyme site sequence of sfi, EcoP151 and BsgI.
2) cofactor: 5 '-gacguacguagcuaggccuauucggccaaaaaaaaaaaaaaaaaa-3'(SEQIDNO: 2)
3) Oligo Mdification primer: 5'-GCCCAGCCAATCACCTAAAGTCA
ggccgaggcggccnNNNNNN'N'N'-3(SEQIDNO:7), wherein NNNNNNN'N'N' is random primer, and Oligo Mdification primer 3 ' holds outermost three continuous bases by two deoxidation moditied processing; Underscore is the restriction enzyme site sequence of sfi.
4) anchor primer: 5'-GCCCAGCCAATCACCTAA-3'(SEQIDNO:5)
2.2 full-length cDNA nucleic acid linear amplification methods
(1) separation and purification of mRNA: get 4 centrifuge tubes, often 10pg total serum IgE got by pipe, 60 DEG C of sex change 5 minutes, every Guan Jun adds OligodT adapter-primer, adding SSC solution to final concentration is 0.5X, mixing solutions room temperature is placed annealing 5 minutes, adds magnetic bead (StreptavidineMagBead) 10 microlitre of affine Streptomycin sulphate, room temperature reaction 8 minutes, collect 20 seconds on magnetic bead frame, abandon supernatant, wash magnetic bead with 0.5X and 0.1XSSC respectively, collect the mRNA that magnetic bead obtains purifying, carry out next step experiment.
The first chain cDNA that (2) 5 ' terminal sequences are known synthesizes: have to collection in 4 side reaction pipes of magnetic bead and add reverse transcription solution and cofactor respectively, 37 DEG C of reactions, 60 minutes (reverse transcription reaction system is in table 4), obtain mRNA-cDNA heterozygosis chain, after reverse transcription terminates, in every side reaction pipe, add 10URNaseH react 8 minutes, utilize enrichment with magnetic bead, remove supernatant, obtain cDNA.
Table 4 reverse transcription reaction system
The cDNA3' end that (3) 5 ' terminal sequences are known extends: in the cDNA that 5 ' terminal sequence of previous step acquisition is known, add Oligo Mdification primer, 68 DEG C of sex change, are slowly annealed to room temperature; In reaction tubes, add PCR reaction soln (reaction system is in table 2), 37 DEG C extend the 3' end of the first chain cDNA, obtain the object cDNA that two terminal sequences are known for 10 minutes.
(4) total length first chain cDNA enriching and purifying: by DEG C sex change of the object cDNA70 in reaction tubes, is placed in reaction tubes on magnetic bead frame after sex change and collects magnetic bead, remove supernatant, obtains the total length first chain cDNA after purifying.
(5) round pcr synthesis amplifying doulbe-chain cDNA: add pcr amplification primer respectively in 4 side reaction pipes, use high-fidelity DNA long fragment polysaccharase to carry out PCR reaction, PCR reaction system is in table 3; PCR reaction conditions is as follows: 95 DEG C of sex change in 5 minutes, 16 to 26 circulations, and cycling condition is as follows: 95 DEG C 20 seconds, 65 DEG C 30 seconds, 72 DEG C 3 minutes.
2.3 experiment conclusion
The amplified production got in 4 arms carries out electrophoretic analysis, and electrophoresis result is shown in Fig. 3.The full-length cDNA nucleic acid linear amplification method of the present invention that shows placenta cDNA amplification can increase the trace mrna template of animal-origin, and the cDNA product total length of amplification experiment and repeatability fine.
Synthesis and the linear amplification of the micro-cDNA of embodiment 3 react
1. the preparation of total serum IgE
Gather turbellarian worm sample, Penaeus vannamei sample, clam sample, aloe sample, Chinese tallow tree seed specimen, use liquid nitrogen grinding powder respectively, five kinds of samples are respectively got 100mg powder and are used Trizol method (illustrating see Invitation Products handbook) to extract each sample total serum IgE.
2. experimental technique
2.1 primer synthesis
1) OligodT adapter-primer: 5 '-Biotin-GCCCAGCCAATCACCTAAAGTCAA
ggccGAGGCggccAGCAGTgcagtTTTTTTTTTTTTTTTTTTVN-3'(SEQIDNO:6), wherein underscore is the restriction enzyme site sequence of sfi, EcoP151 and BsgI.
2) cofactor: 5'-cgacguacguagcuagacuuaucguaaaaaaaaaaaaaaaaaa-3'(SEQIDN O:1), wherein underscore is the restriction enzyme site of RsaI.
3) Oligo Mdification primer: 5'-GCCCAGCCAATCACCTAAAGTCANNNNNNNNN'-3(SEQIDNO:4), wherein NNNNNNNNN' is random primer, and Oligo Mdification primer 3 ' holds outermost base by amination moditied processing.
4) anchor primer: 5'-GCCCAGCCAATCACCTAA-3'(SEQIDNO:5)
2.2 full-length cDNA nucleic acid linear amplification methods
(1) mRNA separation and purification: get 5 little centrifuge tubes, often prop up in centrifuge tube the total serum IgE 10ng adding a kind of sample, 72 DEG C of sex change 3 minutes, add specially designed OligodT adapter-primer, adding SSC solution to final concentration is 0.5X, mixing solutions room temperature is placed annealing 8 minutes, add magnetic bead (StreptavidineMagBead) 10 microlitre of affine Streptomycin sulphate, room temperature reaction 20 minutes, collect 25 seconds on magnetic bead frame, 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.
The first chain cDNA that (2) 5 ' terminal sequences are known synthesizes: have to collection in 5 side reaction pipes of magnetic bead and add reverse transcription solution and cofactor respectively, 45 DEG C of reactions, 90 minutes (reverse transcription reaction system is in table 5), obtain mRNA-cDNA heterozygosis chain, after reverse transcription terminates, in reaction tubes, add 10URNaseH respectively react 5 minutes, 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 often kind of sample of previous step acquisition, add Oligo Mdification primer respectively, 75 DEG C of sex change, slowly be annealed to room temperature (room temperature is specially 20 DEG C), PCR reaction soln (reaction system is in table 2) is added in reaction tubes, 37 DEG C extend the 3' end of cDNA for 10 minutes, obtain the first chain cDNA that two terminal sequences of five kinds of samples are known.
(4) cDNA enriching and purifying: by DEG C sex change of the object cDNA60 in each reaction tubes, is placed in reaction tubes on magnetic bead frame after sex change and collects magnetic bead, remove supernatant, obtains the full-length cDNA of each sample after purifying.
(5) round pcr synthesis amplifying doulbe-chain cDNA: often prop up in centrifuge tube and add anchor primer, use high-fidelity DNA long fragment polysaccharase to carry out PCR reaction, PCR reaction system is in table 3; PCR reaction conditions is as follows: 95 DEG C of sex change in 5 minutes, 16 to 26 circulations, and cycling condition is as follows: 95 DEG C 20 seconds, 65 DEG C 30 seconds, 72 DEG C 3 minutes.
(6) PCR primer of five kinds of samples previous step obtained respectively is 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, amplification method of the present invention is utilized to carry out cDNA amplification to the sample of different sources, the results show, method of the present invention to plant Chinese tallow tree, aloe, marine organisms clam, prawn, and the different plant species such as the biological turbellarian worm in inland lake RNA test all have good amplification, 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 (5)
1. a cDNA nucleic acid amplification method, the steps include:
(1) mRNA separation and purification; Described step (1) is specially: get appropriate total serum IgE or cell pyrolysis liquid in reaction tubes, heat denatured, adds biotin labeled OligodT adapter-primer after annealing; The magnetic bead adding affine Streptomycin sulphate mark in reaction tubes reacts, and collects the mRNA that magnetic bead obtains purifying; Described OligodT adapter-primer length is 25 ~ 80bp, and its sequence comprises left end joint sequence, oligomerization thymus pyrimidine sequence successively from 5 ' end to 3 ' extreme direction, and degenerate core nucleotide sequence VN;
(2) the first chain cDNA that reverse transcription synthesis 5 ' terminal sequence is known; Described step (2) is specially: have to collection in the reaction tubes of magnetic bead and add reagent needed for reverse transcription and carry out reverse transcription reaction, and reaction terminates rear degraded mRNA and collects magnetic bead, the first chain cDNA that 5 ' terminal sequence of acquisition purifying is known; The reverse transcription reaction of described step (2) also adds cofactor, and described cofactor sequence is as shown in SEQIDNO:1 or 2;
The first chain cDNA3' end that (3) 5 ' terminal sequences are known extends, and obtains total length first chain cDNA; Described step (3) is specially: add Oligo Mdification primer in the first chain cDNA that the 5 ' terminal sequence obtained to step (2) is known, sex change, after annealing, add the required reagent of PCR reaction and carry out cDNA3' end extension, obtain two terminal sequences known total length first chain cDNA; 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 the multiple base of continuous print modified;
(4) total length first chain cDNA enriching and purifying; Described step (4) is specially: the reaction solution heat denatured obtained previous step, collects the total length first chain cDNA that magnetic bead obtains purifying;
(5) synthesis of double-strand cDNA or transcribing of RNA; Described step (5) is specially: by method or the in-vitro transcription method of PCR, and the total length first chain cDNA obtained with previous step is for template, and design anchor primer, synthesizes the total length double-stranded DNA that two terminal sequences are known; Or the total length first chain cDNA of the purifying obtained with previous step is for template, design anchor primer, synthesis dsDNA, carries out in-vitro transcription Reactive Synthesis RNA after purifying.
2. nucleic acid amplification method as claimed in claim 1, it is characterized in that, described OligodT adapter-primer sequence is as shown in SEQIDNO:3 or 6.
3. nucleic acid amplification method as claimed in claim 1, it is characterized in that, described Oligo Mdification primer sequence is as shown in SEQIDNO:4 or 7.
4. a cDNA nucleic acid amplification kit, is characterized in that, described test kit comprises and is present in OligodT adapter-primer as shown in SEQIDNO:3 or 6 of sequence in container and the Oligo Mdification primer of sequence as shown in SEQIDNO:4 or 7; Also comprise the cofactor of sequence as shown in SEQIDNO:1 or 2.
5. the application of test kit as described in amplification method as described in claim as arbitrary in claim 1-3 or claim 4 in eukaryote cDNA increases.
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| 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 |
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| 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 |
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