CN101514353A - Clone method of SGAE label 3' end cDNA segment - Google Patents

Clone method of SGAE label 3' end cDNA segment Download PDF

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CN101514353A
CN101514353A CNA2009100463549A CN200910046354A CN101514353A CN 101514353 A CN101514353 A CN 101514353A CN A2009100463549 A CNA2009100463549 A CN A2009100463549A CN 200910046354 A CN200910046354 A CN 200910046354A CN 101514353 A CN101514353 A CN 101514353A
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primer
label
cdna
sgae
pcr
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乔中东
徐汪节
李巧丽
王朝霞
史节平
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Shanghai Jiaotong University
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Shanghai Jiaotong University
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Abstract

The invention relates to a clone method of SGAE label 3' end cDNA segment, belonging to the field of biotechnology. The method comprises the following steps: reverse transcription is carried out on all mRMAs by utilizing the decorated oligo(dT16), 3-5 cytimidine basic groups are added the 3' end of a first cDNA which is synthesized by murine reverse transcriptase, complementary strand of the first cDNA is synthesized by primers, a pair of primers are synthesized according to the known sequence at the two ends of the cDNA for PCR augmentation, and all the cDNAs are concentrated; the cDNA concentrated in the step one is taken as a template, specific segments containing tag are cloned by adopting semi-nested PCR. By adopting the invention, the specific segments containing tag label can be more easily obtained, the process is simpler and the cost is lower.

Description

The cloning process of SGAE label 3 ' end cDNA segment
Technical field
The present invention relates to a kind of segmental cloning process of cDNA of biological technical field, specifically is the cloning process of a kind of SGAE (serial analysis of gene expression) label 3 ' end cDNA segment.
Background technology
Serial analysis of gene expression (SAGE, serial analysis of gene expression) technology is proposed in nineteen ninety-five at first by Velculesce etc.The ultimate principle of genetic expression series is: the nucleotide fragments of per in theory 9 bases can be represented a kind of distinguished sequence of transcription product.SAGE is the method for carrying out the research transcript mRNA of qualitative, quantitative on the full genomic level of a kind of high-throughput, specifically be that fragment in the specific position of every kind of transcript mRNA intercepting 14bp length is the label of SAGE, represent this transcript cDNA sequence with this, order-checking then is one another in series all SAGE labels, analyze its kind and abundance, and then the analysis transcript of qualitative, quantitative.Yet because the only long 14bp fragment of label that SAGE produced, can't carry out sequential analysis to the transcript mRNA of representative, especially the research to the new gene of some unknown SAGE label representatives has very big restriction, influence the further analysis of SAGE data, become a bottleneck of SAGE widespread use.Generally speaking, the thinking of analyzing the SAGE database label is: obtain its downstream 3 ' end cDNA segment from the short-movie section of 14bp, thereby be convenient to obtain its upstream 5 ' end cDNA segment, and then splicing obtains the full length sequence of gene, and obtaining its downstream 3 ' end cDNA segment from the short-movie section of this 14bp is a difficult point.
Find through literature search prior art, Chen, J.J. wait the people to deliver and be entitled as " the Generation of longer cDNA fragments from serial analysis ofgene expression tags for gene identification " paper of (being used for the gene identification method) from the long cDNA fragment of SAGE label generation at " Proceedings of theNational Academy of Sciences " (PNAS) 2000 97 phase 349-353 page or leaf, set forth in the literary composition, utilize wherein antisense primer of an anchored oligo (dT) (grappling oligodeoxynucleotide thymus pyrimidine) conduct, simultaneously with containing the sequence of SAGE tag label as sense primer, under archaeal dna polymerase (Pfu Taq) effect of high-fidelity, carry out the PCR reaction and amplify specific band, but this method has faced following problem: need Initial R NA amount many, the low abundance table label efficient up to standard of amplification is low, it is many that non-specific band appears in amplification, and process is more loaded down with trivial details.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of cloning process of SGAE label 3 ' end cDNA segment is provided.The present invention is more prone to obtain to contain the specific fragment of tag label, and process is simpler, and expense is cheaper.
The present invention is achieved by the following technical solutions, the present invention relates to a kind of cloning process of SGAE label 3 ' end cDNA segment, comprises the steps:
Step 1 is utilized the oligo (dT that modifies 16) all mRMA of reverse transcription, utilize murine reverse transcriptase to add 3-5 cytosine(Cyt) base then, utilize the complementary strand of the synthetic article one cDNA of primer, according to the synthetic a pair of primer of the known array at cDNA two ends at the 3 ' end of synthetic article one cDNA, carry out pcr amplification, the cDNA that enrichment is all;
Step 2, the cDNA that obtains with the step 1 enrichment is a template, adopts the heminested PCR clone to contain the specific fragment of tag.
In the step 1, described modification is specially, at oligo (dT 16) 5 ' end add sequence A AG CAG TGGTAT CAA CGC AGA GTA C, 2 bases of 3 ' least significant end are that VN annexs base.
In the step 1, described primer is specially, 5 '-AAG CAG TGG TAT CAA CGC AGA GTA CGCGGG-3 '.
In the step 1, described known array is specially,
The modified oligo (dT) primer:5 '-AAG CAG TGG TAT CAA CGC AGA GTAC (T) 16VN-3 ', N=A wherein, C, G or T, V=A, G or C;
5′-cap?Oligonucleotide：5′-AAG?CAG?TGG?TAT?CAA?CGC?AGA?GTA?CGCGGG-3′。
In the step 1, described a pair of primer is specially:
PLF：5′-AAG?CAG?TGG?TAT?CAA?CGC?AGA?GT-3′；
PLR：5′-CCA?GAC?ACT?ATG?CTC?ATA?CGA?CG-3′。
In the step 2, described heminested PCR clone is specially: design upstream primer and downstream nested primer, and the specific fragment that contains tag by the twice PCR clone is cloned, and wherein the annealing temperature of PCR is 53 ℃～55 ℃ first, 15～20cycles; The annealing temperature of PCR is 60 ℃ for the second time.
In the step 2, described upstream primer is specially: 5 '-GGATCC+SAGE tag sequence.
In the step 2, described downstream nested primer is specially:
universal?primer?I：5′-CCA?GAC?ACT?ATG?CTC?ATA-3’，
universal?primer?II：5′-CAC?TAT?GCT?CAT?ACG?ACG?CAG?T-3’。
The present invention is at first according to the RACE technology, in the reverse transcription reaction of mRNA, add the preceding paragraph known array at the two ends of all cDNAs, and a pair of primer of sequences Design carries out pcr amplification in view of the above, make all cDNAs obtain increasing to form a large amount of templates and be convenient to subsequent experimental; And then according to the heminested PCR principle, the nested primer of 3 ' the end nested type of design cDNAs pcr template, amplifies the specificity purpose band that contains the SAGE label with the cDNAs that amplifies by twice PCR.
The present invention has following beneficial effect: the present invention has adopted grappling oligo (dT 16) alternative common oligo (dT 16), make reverse transcription product length homogeneous, the present invention total cDNAs that at first increases, overcome the low difficult problem of sample mRNA original bulk, making has enough cDNAs template amounts to be used for follow-up experiment, and especially the clone of the gene of expressing for some low abundance has important effect, for short SAGE label, adopt nested primer, utilize two step heminested PCRs, be more prone to obtain to contain the specific fragment of tag label; With respect to additive method, process of the present invention is simpler, and expense is cheaper.
Description of drawings
Fig. 1 is the synoptic diagram of 3 ' end cDNA segment method (TSAT-PCR) technology mechanism of total cDNAs amplification and two-step pcr amplification SAGE label;
Fig. 2 is the overlapping relation synoptic diagram between used each primer in 3 ' the end cDNA segment method process of two-step pcr amplification SAGE label;
Fig. 3 is total cDNAs amplification electrophoresis proof diagram;
Fig. 4 is the electrophorogram of 16 SGAE labels of 3 ' end cDNA segment method amplification of two-step pcr amplification SAGE label;
Fig. 5 is used for gene identification method comparison diagram for 3 ' end cDNA segment method of two-step pcr amplification SAGE label with from the long cDNA fragment of SAGE label generation.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The implementation method of unreceipted actual conditions in the following example, usually according to normal condition, molecular cloning that the people shows such as Sambrook for example: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1998) condition described in, or the condition of advising according to manufacturer.
Fig. 1 is the explanation of total cDNAs amplification and 3 ' end cDNA segment method (TAST-PCR) technology mechanism, and among the figure, figure A is the amplification of cDNAs, at first with the grappling oligo (dT that is added with one section known array 16) all mRMAs of reverse transcription, utilizing this reversed transcriptive enzyme terminal enzyme (DNA) characteristic then, hold at 5 ' of synthetic article one cDNAs to add 3-5 G, utilize the second chain of these several G synthetic cDNAs under primer 5 '-cap oligonucleotides effect; Utilize all cDNAs two ends all to have common sequence design PLF and a pair of primer of PLR, the total cDNAs of pcr amplification; Figure B is that 3 ' end cDNA segment method (TAST-PCR) amplification of two-step pcr amplification SAGE label contains SAGE label specific fragment synoptic diagram, in the PCR reaction for the first time, the length universal primer I universal primer I (UP-I) of the tag-specific primers of 20bp length and 18bp is the 3 ' end fragment that 55 ℃ of following enrichments contain special label for a pair of primer in annealing temperature; Among the PCR, the universal primer II universalprimer II (UP-II) that signs Auele Specific Primer and 22bp length in 20bp length target is a pair of primer, is to obtain higher 3 ' the cDNAs fragment that contains label of specificity under 60 ℃ in annealing temperature for the second time.
Fig. 2 is the overlapping relation synoptic diagram between used each primer in 3 ' the end cDNA segment method process of two-step pcr amplification SAGE label.
Step 1, the design primer
(1) according to the corresponding primer amplification cDNAs of RACE principle design template
Grappling reverse transcription primer:
the?modified?oligo(dT)primer：
5′-AAG?CAG?TGG?TAT?CAA?CGC?AGA?GTA?C(T) 16VN-3′
(N＝A，C，G?or?T；V＝A，G?or?C)
Synthetic cDNAs second chain 5 ' end primer:
the?5′-cap?oligo?primer：5′-AAG?CAG?TGG?TAT?CAA?CGC?AGA?GTA?CGCGGG-3′；
The a pair of primer of amplification cDNAs:
The Primary Library Forward (PLF) primer:5 '-AAG CAG TGG TAT CAACGC AGA GT-3 ' and
the?Primary?Library?Reverse(PLR)primer：5′-CCA?GAC?ACT?ATG?CTCATA?CGA?CG-3′。
(2) according to the nest-type PRC principle, design sleeve type PCR primer
The upstream primer of twice PCR is tag-specific primers:
The tag-specific primer:5 '-GGATCC+SAGE tag sequence;
The downstream is based on the primer of two nested types of grappling reverse transcription design of primers:
Universal primer I (UP-I): 5 '-CCA GAC ACT ATG CTC ATA-3 ' and
universal?primer?II(UP-II)：5′-CAC?TAT?GCT?CAT?ACG?ACG?CAG?T-3’。
Step 2, the clone of 3 ' terminal sequence of the amplification of cDNAs template and SAGE label
(1) extraction of RNA and RT-PCR
Use Trizol RNA isolation reagent (Invitrogen, Carlsbad, CA, the USA U.S., Ka Ersiba (California), Ying Jun company) from human sperm's cell, to extract total RNA, and pass through content and the purity of UV Detection and Extraction RNA.In the RT-PCR reaction system, utilize the PrimeScript TMReverseTranscriptase (TaKaRa, Dalian, China China, Dalian, TaKaRa company) cultivated 1 hour at 42 ℃, add 2 primers simultaneously: reverse transcription primer the modifiedoligo (dT) primer of article one chain of synthetic cDNA and second strand primer the 5 '-cap oligo primer of synthetic cDNA.
(2) amplification of cDNAs template
As total cNAs template, utilize two ends primer PLF and PLR with above-mentioned RT-PCR reaction solution, under the effect of TakaraEx Taq Hot Start enzyme, total cDNAs template increases; Concrete PCR reaction conditions is: 94 ℃ of 4min; 94 ℃ of 30s, 66 ℃ of 30s, 25 circulations of 72 ℃ of 3min; Final step is extended 5min for 72 ℃; Reaction system is 50ul, specifically sees Table 1.Reaction is got the 10ul reaction solution after finishing, and 1.2% agarose gel electrophoresis detects.
Table 1
Figure A20091004635400081
(3) amplification of specificity sheet degree
The cDNAs that utilizes above-mentioned amplification adopts nested primers UP-I and UP-II as template, and upstream primer is The tag-specific primers, carries out 2 step PCR reactions and amplifies specific band.
PCR reaction for the first time, with primer UP-I and The tag-specific primers, contain 3 ' cDNA fragment of tags in the lower annealing temperature enrichment of Takara Ex Taq enzyme, concrete PCR reaction conditions is: 94 ℃ of 30s, 53 ℃ of-55 ℃ of 30s, 15 circulations of 72 ℃ of 30s; PCR reaction for the second time, with primer UP-II and Thetag-specific primers, contain 3 ' cDNA fragment of tags in 60 ℃ of annealing temperature enrichments of Takara Ex Taq enzyme, concrete PCR reaction conditions is: 94 ℃ of 30s, 60 ℃ of 30s, 25 circulations of 72 ℃ of 30s; The agarose gel electrophoresis of sampling 5ul 1.5% detects.
Implementation result of the present invention detects by following experiment and is confirmed:
(1) confirmation of the amplification of DNAs template
Fig. 3 is sperm cDNAs amplification electrophoresis proof diagram, and gel electrophoresis confirms, presents the smear band of 100bp to 2kb behind the cDNAs template amplification, wherein mainly concentrates on the interval of 300bp to 1kb, and the distributed areas of the length of these and most gene are identical.Among Fig. 3, wherein 1 is the smear of the total cDNAs amplification of sperm; 2 GAPDH genes for contrast.
(2) augmentation detection of specificity sheet degree
Fig. 4 is the electrophorogram of 16 SGAE labels in the SAGE storehouse of 3 ' end cDNA segment method (TAST-PCR) amplification sperm mRNA of two-step pcr amplification SAGE label, and wherein 1-11 is unknown SAGE label, a-e be known SAGE label in contrast; Select label 4 for use, a and e compare for low abundance label.As shown in Figure 4, these fragments are carried out the cloning and sequencing result show that the fragment of 16 label tags of amplification is clear, wherein label 4, a, the label that e expresses for low abundance.Except the small segment of No. 10 labels (this fragment is formed by single primer amplification), all the other fragments all contain the sequence of one section oligo (dT16), and include sequence label, and these features confirm that these sequences are from the 3 ' cDNA fragment corresponding to label.
The effect of present embodiment: with produce long cDNA fragment from the SAGE label and be used for comparing of gene identification method (GLGI) method, present embodiment utilizes grappling reverse transcription primer to synthesize cDNA article one chain, avoid in RT-PCR initial the transcribing of ploy (A) afterbody random order point, thereby cause segmental length to differ, influence the specificity of band.Fig. 5 produces the comparison that long cDNA fragment is used for gene identification method (GLGI) method for 3 ' end cDNA segment method (TAST-PCR) of two-step pcr amplification SAGE label with from the SAGE label.Select for use in the SAGE storehouse of mRNA of sperm label 10,4, A, B, C, E as research object, wherein label 4 and the E label of expressing for low abundance.Find that relatively 3 ' end cDNA segment method (TAST-PCR) of two-step pcr amplification SAGE label amplifies that fragment is comparatively special, especially the label cloning efficiency of expressing in some low abundance is higher, and specificity is better.As shown in Figure 5, contrast and experiment shows, the band of the TAST method amplification of present embodiment is comparatively clear, non-specific amplification is less, and low abundance express tag amplified aspect, comparatively outstanding, can obtain the band of the lower label of abundance, some low-abundance labels and the GLGI method relatively is difficult to increase.Therefore, method of the present invention can be better from the tag amplified 3 ' cDNA fragment that goes out its correspondence of SAGE, especially tag amplified its long segment of expressing for some low abundance is more effective.

Claims (8)

1, a kind of cloning process of SGAE label 3 ' end cDNA segment is characterized in that, comprises the steps:
Step 1 is utilized the oligo (dT that modifies 16) all mRMA of reverse transcription, utilize murine reverse transcriptase to add 3-5 cytosine(Cyt) base then, utilize the complementary strand of the synthetic article one cDNA of primer, according to the synthetic a pair of primer of the known array at cDNA two ends at the 3 ' end of synthetic article one cDNA, carry out pcr amplification, the cDNA that enrichment is all;
Step 2, the cDNA that obtains with the step 1 enrichment is a template, adopts the heminested PCR clone to contain the specific fragment of tag.
According to the cloning process of right 1 described SGAE label 3 ' end cDNA segment, it is characterized in that 2, in the step 1, described modification is specially, at oligo (dT 16) 5 ' end add sequence A AG CAG TGG TATCAA CGC AGA GTA C, 2 bases of 3 ' least significant end are that VN annexs base.
According to the cloning process of right 1 described SGAE label 3 ' end cDNA segment, it is characterized in that 3, in the step 1, described primer is specially, 5 '-AAG CAG TGG TAT CAA CGC AGA GTA CGCGGG-3 '.
According to the cloning process of right 1 described SGAE label 3 ' end cDNA segment, it is characterized in that 4, in the step 1, described known array is specially,
The modified oligo (dT) primer:5 '-AAG CAG TGG TAT CAA CGC AGA GTAC (T) 16VN-3 ', N=A wherein, C, G or T, V=A, G or C;
5′-cap?Oligonucleotide：5′-AAG?CAG?TGG?TAT?CAA?CGC?AGA?GTA?CGCGGG-3′。
According to the cloning process of right 1 described SGAE label 3 ' end cDNA segment, it is characterized in that 5, in the step 1, described a pair of primer is specially:
PLF：5′-AAG?CAG?TGG?TAT?CAA?CGC?AGA?GT-3′；
PLR：5′-CCA?GAC?ACT?ATG?CTC?ATA?CGA?CG-3′。
6, according to the cloning process of right 1 described SGAE label 3 ' end cDNA segment, it is characterized in that, in the step 2, described heminested PCR clone is specially: design upstream primer and downstream nested primer, the specific fragment clone who contains tag by the twice PCR clone, wherein the annealing temperature of PCR is 53 ℃～55 ℃ first, 15～20cycles; The annealing temperature of PCR is 60 ℃ for the second time.
According to the cloning process of right 6 described SGAE label 3 ' end cDNA segments, it is characterized in that 7, in the step 2, described upstream primer is specially: 5 '-GGATCC+SAGE tag sequence.
According to the cloning process of right 6 described SGAE label 3 ' end cDNA segments, it is characterized in that 8, in the step 2, described downstream nested primer is specially:
universal?primer?I：5′-CCA?GAC?ACT?ATG?CTC?ATA-3’，
universal?primer?II：5′-CAC?TAT?GCT?CAT?ACG?ACG?CAG?T-3’。
CNA2009100463549A 2009-02-19 2009-02-19 Clone method of SGAE label 3' end cDNA segment Pending CN101514353A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104955416A (en) * 2012-12-27 2015-09-30 钛格兰技术有限公司 Dental implant unit
CN106636074A (en) * 2017-02-23 2017-05-10 厦门大学 3'RACE (rapid amplification of cDNA ends) method for acquiring complete 3' end sequence as 3' end having repetitive sequence

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104955416A (en) * 2012-12-27 2015-09-30 钛格兰技术有限公司 Dental implant unit
CN106636074A (en) * 2017-02-23 2017-05-10 厦门大学 3'RACE (rapid amplification of cDNA ends) method for acquiring complete 3' end sequence as 3' end having repetitive sequence
CN106636074B (en) * 2017-02-23 2019-07-23 厦门大学 For 3 ' ends with 3 ' the RACE methods for obtaining complete 3 ' end sequence in the case where A repetitive sequence

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