CN101659974A - Method for connecting DNA fragments by utilizing vaccinia virus DNA topoisomerase I - Google Patents
Method for connecting DNA fragments by utilizing vaccinia virus DNA topoisomerase I Download PDFInfo
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- CN101659974A CN101659974A CN200910162805A CN200910162805A CN101659974A CN 101659974 A CN101659974 A CN 101659974A CN 200910162805 A CN200910162805 A CN 200910162805A CN 200910162805 A CN200910162805 A CN 200910162805A CN 101659974 A CN101659974 A CN 101659974A
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Abstract
The invention relates to a method for connecting DNA fragments by utilizing vaccinia virus DNA topoisomerase I. The recognition sequence of the vaccinia virus DNA topoisomerase I is introduced into the design of primer; and after being amplified, the DNA fragments are connected by digestion and/or connecting function of the vaccinia virus DNA topoisomerase I. Furthermore, the method is used for constructing TA, UA, blunt end cloning and directional cloning vector, and can be applied to a kit, thus being greatly convenient for the user and providing good technical platform for studies such as gene cloning, expression, protein function and the like.
Description
Technical field
The present invention relates to a kind of method of utilizing vaccinia virus DNA topoisomerase I (DNA TopoisomeraseI) to connect dna fragmentation.
Background technology
The tradition gene clone method utilizes the goal gene that the method for T4DNA ligase enzyme (T4DNA Ligase) will be to be cloned to be connected with carrier, this recon is transformed in the recipient bacterium clone that Screening and Identification is correct.But the step that this method relates to is many, and loaded down with trivial details time-consuming.Also have a kind of cutting and linkage function of utilizing vaccinia virus DNA topoisomerase I at present, connect the method for dna fragmentation.Vaccinia virus DNA topoisomerase I identification CCCTT, TCCTT, CCCUU, TCCUU, CCCTU, TCCTU site.Invitrogen company has utilized the characteristic in vaccinia virus DNA topoisomerase I identification CCCTT site, cuts through DNA topoisomerase I enzyme, forms different sticky ends, is successfully applied to TA, flush end clone, and directed cloning.This method is compared with traditional method, has fast the characteristics that efficient is high.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of vaccinia virus DNA topoisomerase I (DNA Topoisomerase I) that utilizes can discern CCCTT, TCCTT, CCCUU, TCCUU, CCCTU, the TCCTU site also has enzyme simultaneously and cuts characteristics with linkage function, in order to connecting dna fragmentation, and is applied to the novel method of TA, UA, flush end clone and directed cloning vector construction.With this method generate a reagent box, will make things convenient for the user greatly, provide the good technical platform for realizing researchs such as gene clone, expression, protein function.
The present invention is by the following technical solutions:
The invention provides a kind of method of utilizing vaccinia virus DNA topoisomerase I to connect dna fragmentation, and be used for the novel method of TA, UA, flush end clone and directed cloning vector construction, concrete:
A kind of method of utilizing vaccinia virus DNA topoisomerase I to connect dna fragmentation, this method be by design of primers, utilizes the enzyme of vaccinia virus DNA topoisomerase I to cut with ligation and realize being connected of dna fragmentation, and its step comprises:
(1) design primer: at least two dna fragmentations to be connected are designed primer respectively, every fragment to be connected designs forward primer and reverse primer, 5 ' to 3 ' end of the forward primer of the reverse primer of the last dna fragmentation of adjacent junction correspondence and back one dna fragmentation comprises the linker DNA sequence successively, the complementary dna sequence of vaccinia virus DNA topoisomerase I recognition site and with corresponding template paired dna sequence dna, joint sequence reverse complemental pairing in the forward primer of the reverse primer of wherein said last dna fragmentation and back one dna fragmentation, the acomplementary connector sequence difference of different junctions;
(2) fragment amplification
Be template with corresponding dna fragmentation to be connected respectively, carry out pcr amplification, obtain amplified production with separately primer;
(3) fragment connects
Fragment to be connected is mixed, add vaccinia virus DNA topoisomerase I, 37 ℃ of 15min obtain recombinant dna fragment.
After the described step (2), also comprise the step of pcr amplification product being carried out purifying.
Vaccinia virus DNA topoisomerase I recognition site is XCCYZ in the described step (1), and wherein, X is T or C; Y is T or U; Z is T or U.
In the described step (1), in the design of primers process, the linker DNA sequence that comprises in the primer is at least 3 Nucleotide.
Advantage of the present invention: traditional dna fragmentation method of attachment, need utilize restriction enzyme to make dna fragmentation produce the end that is complementary, utilize T4 ligase enzyme junction fragment behind the purifying.Compare with traditional method, dna fragmentation method of attachment provided by the invention has utilized vaccinia virus DNA topoisomerase I to realize that simultaneously enzyme cuts and the function that is connected, and therefore has characteristics more fast and efficiently; The sequence of to be connected intersegmental part can not exert an influence to connection, eliminated the defective that enzyme in the traditional method is cut step; Complementary joint sequence flexible design is changeable, and connects the specificity height, makes that this method of attachment scope of application is more extensive, particularly is built with remarkable advantages at a plurality of segmental connections.
Description of drawings
Fig. 1: the synoptic diagram that uses the recombinant dna fragment of the inventive method structure;
Wherein: A and B are respectively two dna fragmentations to be connected
C is a recombinant dna fragment
A ' is the amplified production of A, and B ' is the amplified production of B
1 is primer 1 (forward primer of A), and 2 is primer 2 (reverse primer of A)
3 is primer 3 (forward primer of B), and 4 is primer 4 (reverse primer of B)
The electrophorogram that two elements connects among Fig. 2: the embodiment 1;
Wherein: swimming lane M:1Kb Plus DNA Ladder,
3: two segmental connection products of swimming lane.
Embodiment
Material and source used among the embodiment are respectively: plasmid pCMV-cyto-GFP, pcDNA3.1/V5-His/lacZ (American I nvitrogen company), pGL3-Control Vector (Promega company), the TransTaq-T archaeal dna polymerase, 2 * EasyTaq PCR Supermix, Trans2K DNA Marker, Trans2K PlusDNA marker, 1Kb DNA Ladder, 100bp Plus DNA Ladder, Trans DNA Marker IV, pEASY-T3Cloning Kit, EasyPure Plasmid MiniPrep Kit, EasyPure Quick Gel Extraction Kit, the Trans1-T1 competent cell, pUC19 plasmid (Beijing Quanshijin Biotechnology Co., Ltd), primer synthesizes (the handsome Bioisystech Co., Ltd in Beijing).
Embodiment 1:
Connect 2 dna fragmentations that length is 1.2kb with vaccinia virus DNA topoisomerase I.
1.DNA segmental amplification
Design primer according to gene order:
The source of two dna fragmentations, size, primer see Table 1.
The PCR reaction system is 50 μ l.
λ dna profiling (5ng/ μ l) 1 μ l
10 * TransTaq-T Buffer (contains Mg
2+) 5 μ l
TransTaq-T archaeal dna polymerase (5u/ μ l) 0.5 μ l
10mM?dATP 1μl
10mM?dGTP 1μl
10mM?dCTP 1μl
10mM?dTTP 0.5μl
10mM?dUTP 0.5μl
Gene forward primer (10 μ M) 1 μ l
Gene reverse primer (10 μ M) 1 μ l
ddH
2O 37.5μl
Cumulative volume 50 μ l
The PCR reaction conditions is: 94 ℃ of pre-sex change 5 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 1kb/min is in 72 ℃ of extensions, totally 30 circulations; After 72 ℃ of 20 minutes PCR finish, get 5 μ l in 1.5% agarose gel electrophoresis detection.
Utilized dTTP and dUTP in the amplification system simultaneously, product has CCCTT, TCCTT, CCCUU, TCCUU, CCCTU, the recognition site of vaccinia virus DNA topoisomerase Is such as TCCTU.
Table 1PCR amplification the primer, dna fragmentation source and size
Bold-faced letter is represented joint sequence, and underlined letter is represented the complementary sequence of vaccinia virus DNA topoisomerase I (TOPO) recognition site.
2.PCR product purification
Reclaim and purified pcr product with EasyPure Quick Gel Extraction Kit.
3. segmental connection
1) segmental connection
According to steps of processing PCR product:
In the 0.2ml reaction tubes, add:
Vaccinia virus DNA topoisomerase I 1 μ l
Fragment 14 μ l
Fragment 24 μ l
1M Tris-Cl damping fluid, pH 7.5 0.75 μ l
ddH
2O 5.25μl
Cumulative volume 15 μ l
Mix gently, in 37 ℃ of reactions 15 minutes.
2) electrophoresis is confirmed:
In connecting product, add 3 μ l, 6 * Stop Buffer (0.6%SDS, 30mM EDTA pH 8.0) with termination reaction, with fragment 1,2 be connected product and on 1.5% sepharose, carry out electrophoresis.Result such as Fig. 3.
As seen, connect the DNA band that occurs tangible 2.4kb in the product, prove successful connection.
Sequence table
＜110〉Beijing Quanshijin Biotechnology Co., Ltd
＜120〉a kind of method of utilizing vaccinia virus DNA topoisomerase I to connect dna fragmentation
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<141>2009-08-07
<160>4
<210>1
<211>22
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＜213〉artificial sequence
<220>
＜223〉single stranded DNA, PCR prepare the required forward primer of fragment 1.
<400>1
taggctagca?aaggagaaga?ac?22
<210>2
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉single stranded DNA, PCR prepare the required reverse primer of fragment 1, contain the complementary dna sequence of linker DNA sequence and vaccinia virus DNA topoisomerase I recognition site
<400>2
gatcagaagg?actttgtaga?gctcatccat?gc?32
<210>3
<211>32
<212>DNA
＜213〉artificial sequence
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＜223〉single stranded DNA, PCR prepare the required forward primer of fragment 2, contain the complementary dna sequence of linker DNA sequence and vaccinia virus DNA topoisomerase I recognition site
<400>3
ctgatcaagg?gtccgctcat?gagacaataa?cc?32
<210>4
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉single stranded DNA, PCR prepare the required reverse primer of fragment 2.
<400>4
ttaccaatgc?ttaatcagtg?agg?23
Claims (4)
1, a kind of method of utilizing vaccinia virus DNA topoisomerase I to connect dna fragmentation is characterized in that: by design of primers, utilize the enzyme of vaccinia virus DNA topoisomerase I to cut with ligation and realize being connected of dna fragmentation, its step comprises:
(1) design primer: at least two dna fragmentations to be connected are designed primer respectively, every fragment to be connected designs forward primer and reverse primer, 5 ' to 3 ' end of the forward primer of the reverse primer of the last dna fragmentation of adjacent junction correspondence and back one dna fragmentation comprises the linker DNA sequence successively, the complementary dna sequence of vaccinia virus DNA topoisomerase I recognition site and with corresponding template paired dna sequence dna, joint sequence reverse complemental pairing in the forward primer of the reverse primer of wherein said last dna fragmentation and back one dna fragmentation, the acomplementary connector sequence difference of different junctions;
(2) fragment amplification
Be template with corresponding dna fragmentation to be connected respectively, carry out pcr amplification, obtain amplified production with separately primer;
(3) fragment connects
Fragment to be connected is mixed, add vaccinia virus DNA topoisomerase I, 37 ℃ of 15min obtain recombinant dna fragment.
2, the method for utilizing vaccinia virus DNA topoisomerase I to connect dna fragmentation according to claim 1 is characterized in that: after the described step (2), also comprise the step of pcr amplification product being carried out purifying.
3, the method for utilizing vaccinia virus DNA topoisomerase I to connect dna fragmentation according to claim 1, it is characterized in that: vaccinia virus DNA topoisomerase I recognition site is XCCYZ in the described step (1), and wherein, X is T or C; Y is T or U; Z is T or U.
4, the method for utilizing vaccinia virus DNA topoisomerase I to connect dna fragmentation according to claim 1, it is characterized in that: in the described step (1), in the design of primers process, the linker DNA sequence that comprises in the primer is at least 3 Nucleotide.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642829A (en) * | 2013-12-13 | 2014-03-19 | 北京全式金生物技术有限公司 | High-efficiency gene directional cloning method |
| CN106191171A (en) * | 2016-07-27 | 2016-12-07 | 上海毕傲图生物科技有限公司 | A kind of method utilizing T4 phage DNA topoisomerase I to connect DNA fragmentation |
| CN106434719A (en) * | 2016-09-27 | 2017-02-22 | 北京擎科新业生物技术有限公司 | Method of constructing zero-background support based on vaccinia topoisomerase I |
| CN109576254A (en) * | 2019-01-11 | 2019-04-05 | 吴江近岸蛋白质科技有限公司 | The preparation method and expression and purification method of DNA topoisomerase I |
-
2009
- 2009-08-07 CN CN200910162805A patent/CN101659974A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642829A (en) * | 2013-12-13 | 2014-03-19 | 北京全式金生物技术有限公司 | High-efficiency gene directional cloning method |
| CN103642829B (en) * | 2013-12-13 | 2016-05-18 | 北京全式金生物技术有限公司 | A kind of method of high efficiency gene directed cloning |
| CN106191171A (en) * | 2016-07-27 | 2016-12-07 | 上海毕傲图生物科技有限公司 | A kind of method utilizing T4 phage DNA topoisomerase I to connect DNA fragmentation |
| CN106434719A (en) * | 2016-09-27 | 2017-02-22 | 北京擎科新业生物技术有限公司 | Method of constructing zero-background support based on vaccinia topoisomerase I |
| CN109576254A (en) * | 2019-01-11 | 2019-04-05 | 吴江近岸蛋白质科技有限公司 | The preparation method and expression and purification method of DNA topoisomerase I |
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Application publication date: 20100303 |