CN106868005A - A kind of anchor primer and amplification method for efficient Rapid amplification of cDNA ends - Google Patents
A kind of anchor primer and amplification method for efficient Rapid amplification of cDNA ends Download PDFInfo
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Abstract
The present invention discloses a kind of anchor primer for efficient rapid amplifying cDNA5 ' and 3 ' ends, described two anchor primers can specifically bind to target nucleic acid sequence and trigger reverse transcription extension, so as to efficient rapid amplifying goes out the ends of cDNA5 ' and 3 ', 5 ' and 3 ' the end rapid amplifyings of cDNA in low abundance transcript are particularly applicable to.In addition, the present invention also provides a kind of kit and amplification method of efficient rapid amplifying cDNA5 ' and 3 ' ends.
Description
Technical field
It is more particularly to a kind of to be used for efficient rapid amplifying cDNA the present invention relates to a kind of rapid amplifying method of cDNA ends
The anchor primer and amplification method of end.
Background technology
The acquisition of full-length gene is an emphasis of bioengineering and molecular biology research.Classical RACE (rapid-
Amplification of cDNA ends) technology is to be equal to a technology of invention in 1988 by Frohman, the technology can be with
Effectively obtain the full length sequence of gene.RACE is based on RT-PCR technology, and end adds locking primer to be obtained from RNA and carries and end
The cDNA of end locking Primers complementary or identical sequence, then performing PCR amplification is entered so as to obtain complete cDNA by specific primer
5' and 3' terminal sequences.RACE know-whies are simple, and it has the advantages that quick, efficient, cheap.
In many biological studies, the genes of interest abundance studied is often very low, in this case traditional RACE skills
Art just highlights its deficiency, such as poor specificity, and amplification efficiency is low, therefore obtains full-length cDNA change by transcription from low abundance mRNA
Obtain abnormal difficult.Therefore, this area is in the urgent need to exploitation is quick, efficiency high, the specific good RLM-RACE skills of cDNA 5 ' and 3 '
Art.
The content of the invention
There is provided a kind of for efficient rapid amplifying it is an object of the invention to overcome above-mentioned the deficiencies in the prior art part
The anchor primer of cDNA ends, the anchor primer can specifically bind to target nucleic acid sequence and trigger reverse transcription extension,
So as to efficient rapid amplifying goes out the ends of cDNA 5 ' and 3 '.In addition, the present invention also provides a kind of efficient rapid amplifying cDNA ' ends
Kit and amplification method.
To achieve the above object, the technical scheme that the present invention takes is for a kind of for the efficient ends of rapid amplifying cDNA 5 '
Anchor primer, the sequence end of 5 ' end anchor primer contains 3 guanines, and wherein the first two guanine is monodeoxy bird
Purine, a guanine of least significant end is lock nucleic acid modification guanine.
Preferably, the sequence itself of 5 ' end anchor primer forms the stem cyclic structure that 3 ' end sections are protruded.
Preferably, the sequence of 5 ' end anchor primer does not exist specific binding region with all species transcripts.
Preferably, the cytosine base complementary pairing of the sequence end of 5 ' end anchor primer and the ends of cDNA 5 ',
When M-MLV reverse transcriptase synthesizes cDNA to end, extended as template using 5 ' end anchor primer.
Preferably, the sequence of 5 ' end anchor primer is：ATGCAGACGATTCTACGCT GTGTTCTArGrG+G.
Using the sequence of this 5 ' end anchor primer, it is amplifiable go out the one section long full-length cDNA of chain non-coding RNA, its UCSC database
Accession number is TCONS_00027227, and 27227 are named as certainly, its chromosome mapping position:chr19:22025306-
22035178。
On the other hand, the present invention provides a kind of kit of efficient Rapid amplification of cDNA ends, and the kit contains has the right
Profit requires 5 ' the end anchor primers and 3 ' end anchor primers described in 1~5 any one.
Preferably, 3 ' the end grappling primer sequence ends connection in the kit of the efficient Rapid amplification of cDNA ends
30 dT, and 3 ' the end grappling primer sequence itself does not form hairpin structure；And anchor primer sequence and all species
Transcript does not exist specific binding region.
Preferably, 3 ' the end grappling primer sequence is CGATCTGGGCTCTGTGTTCTC- (dT) 30.
On the other hand, the present invention provides a kind of method of efficient rapid amplifying cDNA, the described method comprises the following steps：
(1), the cDNA fragments of the 5' ends of amplifying target genes, obtain the RACE products of 5' ends；(2), the 3' of amplifying target genes
The cDNA fragments of end, obtain the RACE products of 3' ends；(3), 2 obtained from step (1) and step (2) have phase mutual respect
Full-length cDNA is obtained in the RACE products of the 3' and 5' ends of folded sequence, or by analyzing 3' the and 5' terminal sequences of RACE products,
Synthesize corresponding primer and amplify full-length cDNA.
Preferably, the method for the RACE products for obtaining 5' ends in the step (1) is：Using 5 ' end anchor primers as
Sense primer, gene specific primer R-Primer, as anti-sense primer, is template with the first chain cDNA for synthesizing, and enters performing PCR and follows
Ring, target gene 5 ' end cDNA fragment amplifications out, obtain the RACE products of 5' ends；Obtained in the step (2)
The method of the RACE products of 3' ends is：Using gene specific primer F-Primer as sense primer, 3' ends anchor primer is made
It is anti-sense primer, is template with the first chain cDNA for synthesizing, carry out PCR cycle, the cDNA fragments of genes of interest 3' ends is expanded
Increase out, obtain the RACE products of 3' ends.
The guanine of 5 ' end anchor primers is combined with the tight jail of the cytosine base complementary pairing of cDNA 5' ends, works as M-
During the reverse transcription of MLV reverse transcriptase to cDNA ends, M-MLV reverse transcriptase is extended using anchor primer as template, transcription
Contain the sequence complementary with anchor primer in product end；The thymidine of 3' ends anchor primer and the poly of cDNA 3' ends
(A) tail adenine base complementary pairing is combined, and M-MLV reverse transcriptase is extended using anchor primer as template, transcription product
5' ends contain and anchor primer identical sequence.
The principle schematic of the method for efficient rapid amplifying cDNA of the present invention is as shown in figure 1, the present invention is to tradition
RACE technologies carry out improvement optimization, and specificity locking primer is separately designed for cDNA 5' ends and 3' ends.CDNA5' ends
In amplification, M-MLV reverse transcriptase has terminal transferase activity, adds 3- automatically when reverse transcription reaches the 5' ends of the first chain
5 (dC) residues, mix 5' ends containing guanine (dG) specificity locking primer in system, specificity locking primer (dG) with
After cDNA cytimidines (dC) Residue pairing, be converted to specificity locking primer sequence and connect general connecing as template continues extension
Header sequence, using the universal primer UP (universal primer, UP) containing Partial joints sequence as sense primer, gene
Special primer GSP (genespecific primer, GSP), as anti-sense primer, is template with the first chain cDNA for synthesizing, and is entered
Performing PCR circulate, target gene 5 ' end cDNA fragment amplifications out；In cDNA3' RLM-RACEs, using the 3' ends of mRNA
Poly (A) tails at end as a primer binding site, using the sequences of Oligo (dT) 30 of connection universal adapter-primer as lock
Determine primed reverse transcription synthetic standards the first chain cDNA, then drawn as sense primer with a gene specific, portion is contained with one
Divide the sequence complementary with universal joint as anti-sense primer, with the chains of cDNA first as template, PCR cycle is carried out, genes of interest
The DNA fragmentation of 3' ends is amplified and；Finally, have from 2 overlapped sequence 3' and 5' RACE products in obtain total length
CDNA, or by analyzing 3' the and 5' terminal sequences of RACE products, synthesize corresponding primer and amplify full-length cDNA.
The beneficial effects of the present invention are：The present invention be based on M-MLV reverse transcriptase terminal transferase activities, using PCR from
5 ' and 3 ' ends of rapid amplifying cDNA in low abundance transcript, the amplification method has easy to operate, quick, amplification efficiency
The high, advantage that specificity is good, is used especially for the rapid amplifying of 5 ' and 3 ' ends of cDNA from low abundance transcript.
Brief description of the drawings
Fig. 1 is the principle schematic of the method for efficient rapid amplifying cDNA of the present invention, and wherein rG is that monodeoxy bird is fast
Purine (riboguanosine), rG is that lock nucleic acid modifies guanine (LNA)；
Fig. 2 is to expand this by the institute of embodiment 2 using the method for the efficient ends of rapid amplifying cDNA 5 ' and 3 ' of the present invention
5 ' and 3 ' ends of cDNA are stated, then amplified production carries out the electrophoresis detection figure of 1.5% agarose gel electrophoresis, the wherein generation of swimming lane 1
The RLM-RACE product of table 5 ', swimming lane 2 represents 3 ' RLM-RACE products, and swimming lane M represents DL2000marker.
Specific embodiment
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with specific embodiment and accompanying drawing pair
The present invention is described further.
Embodiment 1
A kind of method of the efficient ends of rapid amplifying cDNA 5 ' and 3 ', comprises the following steps：
A. one section of specificity locking primer, i.e. anchor primer being connected with the ends of cDNA 5 ' is designed, LT is designated as.The sequence
3 ' ends, 3 guanines of band, wherein the first two guanine are monodeoxy guanine (riboguanosine), one of least significant end
Guanine base is that lock nucleic acid modifies guanine (LNA), but the primer itself forms the prominent stem cyclic structure of 3 ' ends part.
The special modification of joint sequence end and self structure are remarkably improved the anchoring efficiencies of locking primer；
B. one section of anchor series closed with poly (A) caudal knot of 3' ends is designed, MT is designated as.The sequence end contains 30 chests
Gland pyrimidine, itself does not form hairpin structure；
C. design a primer complementary with MT sequence parts, be designated as B；
D. design obtains two sections of specific primers for target gene, wherein the primer sequence note of amplification cDNA 5 ' ends
It is R-Primer, the primer sequence of amplification cDNA 3 ' ends is designated as F-Primer；
E.cDNA 5' RLM-RACEs：Sample total serum IgE is extracted, total serum IgE and LT anchor primers is placed in and is contained M-MLV reversions
The synthesis of cDNA is carried out in the reverse transcription reaction system for recording enzyme.When carrying out cDNA synthesis using the anchor primer, anchor primer
Guanine and the cytosine base complementary pairing of cDNA 5' ends, M-MLV reverse transcriptase are prolonged using anchor primer as template
Stretch, the sequence complementary with anchor primer is contained in transcription product end.By the use of LT primers as sense primer, gene specific primer R-
Primer is template with the first chain cDNA for synthesizing as anti-sense primer, carry out PCR cycle, target gene 5 ' end
CDNA fragment amplifications are out.
F.cDNA 3' RLM-RACEs：Sample total serum IgE is extracted, total serum IgE and MT anchor primers is placed in and is contained M-MLV reversions
The synthesis of cDNA is carried out in the reverse transcription reaction system for recording enzyme.When carrying out cDNA synthesis using the anchor primer, anchor primer
Poly (A) tail adenine base complementary pairing of thymidine and cDNA3' ends, M-MLV reverse transcriptase using anchor primer as
Template is extended, and the 5' ends of transcription product are contained and anchor primer identical sequence.Using gene specific primer F-
Primer, as anti-sense primer, is template with the first chain cDNA for synthesizing as sense primer, B primers, carries out PCR cycle,
The cDNA fragment amplifications of genes of interest 3' ends are out.
The rapid amplifying of the ends of 2 27227lncRNA of embodiment 5 ' and 3 '
27227lncRNA is long-chain non-coding RNA, does not know function and the gene name of the long-chain non-coding RNA also at present
Claim.The accession number of known its UCSC database is TCONS_00027227,27227 is named as certainly, its chromosome mapping
position:chr19:22025306-22035178。
According to the method amplification 27227lncRNA5 ' and 3 ' ends described in embodiment 1, its key component is：
1st, the design of nucleotide sequence
Specific nucleic acid sequence used in the present embodiment, particular sequence is as follows：
Specific nucleic acid sequence used in the present embodiment of table 1
Wherein, rG is monodeoxy guanine (riboguanosine) in upper table, and+G is that lock nucleic acid modifies guanine (LNA),
Then reverse transcription reaction system is prepared according to table 2：
The reverse transcription reaction system of table 2
After the completion of reverse transcription reaction system is prepared, reverse transcription reaction is carried out according to the circulating system of table 3；
The reverse transcription reaction circulating system of table 3
Next, pcr amplification reaction is carried out, wherein preparing 5' RLM-RACEs reaction system according to table 4, being prepared according to table 5
3' RLM-RACE reaction systems：
The 5' RLM-RACE reaction systems of table 4
The 3' RLM-RACE reaction systems of table 5
After preparation system is finished, upper machine enters performing PCR amplification, and wherein PCR amplification programs are：
After completion of the reaction, the amplified production of the ends of cDNA5 ' and 3 ' is carried out into 1.5% agarose gel electrophoresis detection, as a result such as
Shown in Fig. 2, wherein swimming lane 1 represents 5 ' RLM-RACE products, and swimming lane 2 represents 3 ' RLM-RACE products, clear in this two swimming lanes
Clear visible bright amplified band, product is sequenced after agarose electrophoresis recovery purifying, and sequencing result is compared and 27227lncRNA
Long-chain non-coding RNA sequence is consistent, shows that we successfully obtain the ends of cDNA5 ' and 3 '.
It is last to should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail to the present invention with reference to preferred embodiment, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent, without deviating from the essence of technical solution of the present invention
And scope.
Claims (10)
1. one kind is used for the anchor primer of the efficient ends of rapid amplifying cDNA 5 ', it is characterised in that 5 ' end anchor primer
Sequence end contain 3 guanines, wherein the first two guanine is monodeoxy guanine, and a guanine of least significant end is lock core
Acid modification guanine.
2. the anchor primer of the efficient ends of rapid amplifying cDNA 5 ' is used for as claimed in claim 1, it is characterised in that described
The sequence itself of 5 ' end anchor primers forms the stem cyclic structure that 3 ' end sections are protruded.
3. the anchor primer of the efficient ends of rapid amplifying cDNA 5 ' is used for as claimed in claim 1, it is characterised in that described
The sequence of 5 ' end anchor primers does not exist specific binding region with all species transcripts.
4. the anchor primer of the efficient ends of rapid amplifying cDNA 5 ' is used for as claimed in claim 1, it is characterised in that described
The sequence end of 5 ' end anchor primers and the cytosine base complementary pairing of the ends of cDNA 5 ', treat that M-MLV reverse transcriptase synthesizes
When cDNA is to end, extended as template using 5 ' end anchor primer.
5. the anchor primer for the efficient ends of rapid amplifying cDNA 5 ' as described in any one of Claims 1 to 4, its feature
It is that the sequence of 5 ' end anchor primer is ATGCAGACGATTCTACGCTGTGTTCTArGrG+G.
6. a kind of kit of efficient Rapid amplification of cDNA ends, it is characterised in that the kit contains Claims 1 to 5
Described in any one for the anchor primer of the efficient ends of rapid amplifying cDNA 5 ' and for efficient rapid amplifying cDNA 3 '
The anchor primer of end.
7. a kind of kit of efficient Rapid amplification of cDNA ends as claimed in claim 6, it is characterised in that described for height
The anchor primer sequence end of the ends of effect rapid amplifying cDNA 3 ' connects 30 dT, and 3 ' the end grappling primer sequence
Itself does not form hairpin structure；And anchor primer sequence does not exist specific binding region with all species transcripts.
8. a kind of kit of efficient Rapid amplification of cDNA ends as claimed in claim 6, it is characterised in that described for height
The anchor primer sequence of the ends of effect rapid amplifying cDNA 3 ' is CGATCTGGGCTCTGTGTTCTC- (dT) 30.
9. a kind of method of efficient rapid amplifying cDNA of kit using as described in claim 6-7 is any, it is characterised in that
The described method comprises the following steps：(1) mesh, is expanded using the anchor primer for the efficient ends of rapid amplifying cDNA 5 '
Gene 5' ends cDNA fragments, obtain the RACE products of 5' ends；(2), using for efficient rapid amplifying cDNA3 '
The cDNA fragments of the 3' ends of the anchor primer amplifying target genes of end, obtain the RACE products of 3' ends；(3), from step
(1) 2 for and in step (2) obtaining obtain full-length cDNA in having the RACE products of the 3' and 5' ends of overlapped sequence, or
By analyzing 3' the and 5' terminal sequences of RACE products, synthesize corresponding primer and amplify full-length cDNA.
10. a kind of method of efficient rapid amplifying cDNA as claimed in claim 9, it is characterised in that in the step (1)
Method to the RACE products of 5' ends is：Anchor primer for the efficient ends of rapid amplifying cDNA 5 ' draws as upstream
Thing, genes of interest special primer R-Primer, as anti-sense primer, is template with the first chain cDNA for synthesizing, and carries out PCR cycle,
Target gene 5 ' end cDNA fragment amplifications out, obtain the RACE products of 5' ends；3' ends are obtained in the step (2)
The method of the RACE products at end is：Using genes of interest special primer F-Primer as sense primer, for efficient rapid amplifying
The anchor primer of the ends of cDNA 3 ', as anti-sense primer, is template with the first chain cDNA for synthesizing, and PCR cycle is carried out, purpose
The cDNA fragment amplifications of gene 3' ends out, obtain the RACE products of 3' ends.
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| CN107747134A (en) * | 2017-10-20 | 2018-03-02 | 重庆天科雅生物科技有限公司 | A kind of method for building people TCRalphaCDR3 areas library |
| CN107760678A (en) * | 2017-10-31 | 2018-03-06 | 安徽省农业科学院水产研究所 | The amplification method of 3 ' RACE adapter-primers and 3 ' end unknown gene sequences |
| CN107829145A (en) * | 2017-10-20 | 2018-03-23 | 重庆天科雅生物科技有限公司 | A kind of method for building mouse TCRalphaCDR3 areas library |
| CN107858400A (en) * | 2017-10-20 | 2018-03-30 | 重庆天科雅生物科技有限公司 | A kind of method for building mouse TCRbetaCDR3 areas library |
| CN107893068A (en) * | 2017-10-20 | 2018-04-10 | 重庆天科雅生物科技有限公司 | A kind of method for building people TCRbetaCDR3 areas library |
| CN108624587A (en) * | 2018-05-07 | 2018-10-09 | 西南大学 | The RACE methods of efficient quick obtaining Plant RNA viral end sequence |
| CN110819673A (en) * | 2018-08-07 | 2020-02-21 | 华中农业大学 | Prokaryotic cDNA 5' end rapid amplification method |
| CN113981148A (en) * | 2021-11-18 | 2022-01-28 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Method for identifying RV infection and virus genotype and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107747134A (en) * | 2017-10-20 | 2018-03-02 | 重庆天科雅生物科技有限公司 | A kind of method for building people TCRalphaCDR3 areas library |
| CN107829145A (en) * | 2017-10-20 | 2018-03-23 | 重庆天科雅生物科技有限公司 | A kind of method for building mouse TCRalphaCDR3 areas library |
| CN107858400A (en) * | 2017-10-20 | 2018-03-30 | 重庆天科雅生物科技有限公司 | A kind of method for building mouse TCRbetaCDR3 areas library |
| CN107893068A (en) * | 2017-10-20 | 2018-04-10 | 重庆天科雅生物科技有限公司 | A kind of method for building people TCRbetaCDR3 areas library |
| CN107760678A (en) * | 2017-10-31 | 2018-03-06 | 安徽省农业科学院水产研究所 | The amplification method of 3 ' RACE adapter-primers and 3 ' end unknown gene sequences |
| CN108624587A (en) * | 2018-05-07 | 2018-10-09 | 西南大学 | The RACE methods of efficient quick obtaining Plant RNA viral end sequence |
| CN110819673A (en) * | 2018-08-07 | 2020-02-21 | 华中农业大学 | Prokaryotic cDNA 5' end rapid amplification method |
| CN110819673B (en) * | 2018-08-07 | 2021-07-13 | 华中农业大学 | Prokaryotic cDNA 5' end rapid amplification method |
| CN113981148A (en) * | 2021-11-18 | 2022-01-28 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Method for identifying RV infection and virus genotype and application thereof |
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| CN106868005B (en) | 2020-07-21 |
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