CN107058357A - A kind of TA cloning vectors and its construction method and application based on digestion - Google Patents
A kind of TA cloning vectors and its construction method and application based on digestion Download PDFInfo
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- CN107058357A CN107058357A CN201710147060.XA CN201710147060A CN107058357A CN 107058357 A CN107058357 A CN 107058357A CN 201710147060 A CN201710147060 A CN 201710147060A CN 107058357 A CN107058357 A CN 107058357A
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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Abstract
The invention discloses a kind of TA cloning vectors based on digestion and its construction method and application, the nucleotide sequence of the TA cloning vectors based on digestion is named as pTA03 carriers as shown in SEQ ID NO.1.After the carrier is digested through restriction enzyme A hdI, the linear carrier fragment of purifying can be used for TA to clone, and the carrier carries ccdB lethal genes, be screened without carrying out blue hickie, and positive rate is close to 100%.Program reliability is high, and cost is relatively low, can be prepared on a large scale.
Description
Technical field
The invention belongs to biology field, specifically, it is related to a kind of TA cloning vectors and its structure based on digestion
Construction method and application.
Background technology
TA clones are most easy, quick, the economic methods of current clone PCR products, and pole is applied in biology field
Its is extensive.According to the species of the enzyme used in course of reaction, TA clones can be broadly divided into two classes, and a class is to be based on T4DNA connections
Enzyme a, class is to be based on DNA topoisomerase Is (TOPO), and wherein the former carrier is prepared easily, and cost is low, and efficiently time-consuming for connection,
The latter's carrier prepares cumbersome, cost height, and efficiently connection is time-consuming extremely short.The mode of T ends is produced according to carrier, transferase is utilized
T and digestion is added to produce two kinds of T ends in PCR primer end, the former flux is high, and uniformity is poor, and the latter's flux is lower slightly, uniformity
It is good.
The content of the invention
In view of this, there is provided a kind of TA cloning vectors and its structure based on digestion for the problem of present invention is directed to above-mentioned
Methods and applications, the TA cloning vectors reliability is high, and uniformity is good, and with low cost, middle-size and small-size laboratory is that can be used.
In order to solve the above-mentioned technical problem, it is described to be based on enzyme the invention discloses a kind of TA cloning vectors based on digestion
The nucleotide sequence for the TA cloning vectors cut is named as pTA03 carriers as shown in SEQ ID NO.1.
The present invention also provides a kind of construction method of the TA cloning vectors based on digestion, comprises the following steps：Take 3-5 μ g
PTA03 carriers, are digested 3-18 hours under the conditions of 37 DEG C, digestion is added on a small quantity after terminating with restriction enzyme A hdI
EcoRV-HF endonuclease digestions 30 minutes, 65 DEG C are kept for 20 minutes, and digestion products are directly crossed post using cleavage reagent box and reclaimed, or
Person is reclaimed with DNA gel QIAquick Gel Extraction Kit；- 20 DEG C of preservations of recovery product, the product is the TA cloning vectors prepared.
The present invention also provides a kind of application method of the above-mentioned TA cloning vectors based on digestion, comprises the following steps：
Step 1, TA cloning vector of the preparation based on digestion：Take under the conditions of 3-5 μ g pTA03 carriers, 37 DEG C with restricted
Enzyme cutting AhdI is digested 3-18 hours, and digestion adds a small amount of EcoRV-HF endonuclease digestions 30 minutes after terminating, 65 DEG C of holdings
20 minutes, digestion products were directly crossed post using cleavage reagent box and reclaimed, or are reclaimed with DNA gel QIAquick Gel Extraction Kit；Reclaim production
- 20 DEG C of preservations of thing, the product is the TA cloning vectors prepared；
Step 2, the TA cloning vectors based on digestion that step 1 is prepared are connected instead with purpose PCR fragment mixed preparing
System is answered, of short duration centrifugation 3-5 seconds after mixing react mixed liquor 5 minutes in 22 degree, and reaction end is rearmounted on ice, carried out follow-up
Conversion reaction.
Further, the endonuclease reaction system in step 1 is specially：10xCutSmart Buffer (NEB) 10 μ L, 10U/
μ L restriction enzyme A hdI 2 μ L, 1 μ g/ μ l the μ L of TA cloning vectors 5 based on digestion, surplus is H2O, it is total with upper volume
Measure as 100 μ L.
Further, the coupled reaction system in step 2 is specially：10xT4DNA ligase Buffer (NEB) 1 μ L,
400U/ μ L μ L, the 50ng/ μ L of the T4DNA ligase 0.5 μ L of TA cloning vectors 1 based on digestion, purpose PCR fragment and base
It is 3~7: 1 in the mol ratio of the TA cloning vectors of digestion, surplus is ddH2O, using upper volume total amount as 100 μ L.
Further, the addition of the TA cloning vectors based on digestion in linked system is 0.03pmol, as purpose PCR
The length of fragment is in below 2000bp, purpose PCR fragment：The mol ratio of TA cloning vectors based on digestion is 7: 1,22 DEG C of companies
Connect 5 minutes；When the length of purpose PCR fragment is in more than 2000bp, purpose PCR fragment：The mol ratio of TA cloning vectors is 3:
1,22 DEG C connects 30 minutes.
Further, the consumption (μ l) of purpose PCR fragment=[660 × A × 0.03 × B (ng)] ÷ [1000 × purpose PCR
Fragment concentrations (ng/ μ l)], wherein A is the base logarithm (bp) of purpose PCR fragment, and B is PCR fragment：Mole of TA cloning vectors
Than.
Compared with prior art, the present invention can be obtained including following technique effect：
1) TA cloning vectors of the present invention are prepared simply, and cost is low, and digestion can once prepare a large amount of carriers；
2) TA cloning vectors reliability of the present invention is high, and uniformity is good, the substantially all band T in end；
3) TA cloning vectors of the present invention carry lethal gene, without the screening of blue hickie, and positive rate is close to 100%.
Certainly, any product for implementing the present invention it is not absolutely required to while reaching all the above technique effect.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes the part of the present invention, this hair
Bright schematic description and description is used to explain the present invention, does not constitute inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 is the collection of illustrative plates of the TA cloning vectors of the invention based on digestion；
Fig. 2 is growing state of the positive bacteria of the present invention on LB/Amp plating mediums；
Fig. 3 is different bacterium colony PCR qualification results of the invention, wherein, M is D2000DNA Marker, and 1-6 is random PCR
Identification.
Embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technology effect.
TA cloning vector and its construction method of the embodiment 1 based on digestion
The present invention provides a kind of TA cloning vectors based on digestion, and this carrier is formed by pUC19 carriers for framework construction, no
Containing multiple cloning sites, ccdB lethal genes are carried, positive rate is close to 100%, it is adaptable to DNA sequencing.Using restriction enzyme
After the AhdI digestions carrier, the TA cloning vectors linearized are reclaimed in purifying, are named as pTA03 carriers.The Vector map
As shown in figure 1, the nucleotide sequence of TA cloning vectors is as shown in SEQ ID NO.1, its target fragment sequence is as follows：WhereinIt is M13fwd and M13rev primer sequences respectively,It is AmpR sequences,It is in restricted
Enzyme cutting AhdI sequences, thickened portion is ccdB lethal gene fragments.
The nucleotide sequence of TA cloning vectors is as follows：
The structure and application method of TA cloning vector of the embodiment 2 based on digestion
3-5 μ g pTA03 plasmids are taken, are digested 3-18 hours with restriction enzyme A hdI, digestion is added few after terminating
Measure EcoRV-HF endonuclease digestions 30 minutes, 65 DEG C are kept for 20 minutes, digestion products are directly crossed post using cleavage reagent box and reclaimed,
Also it can be reclaimed with DNA gel QIAquick Gel Extraction Kit.- 20 DEG C of preservations of recovery product, the product is the TA cloning vectors prepared, can
It is connected with carrying out TA with T4DNA ligases and PCR primer, connection product is without blue hickie screening.
Embodiment 3
1. vector linearization：As shown in table 1, digestion system is prepared, 37 DEG C are reacted 3-18 hours, and 1 μ is added in digestion after terminating
L EcoRV-HF restriction endonucleases (NEB) are digested 30 minutes, and 65 DEG C are kept for 20 minutes, and digestion products directly cross post using cleavage reagent box
Reclaim, it is also possible to which DNA gel QIAquick Gel Extraction Kit is reclaimed, -20 DEG C of preservations of recovery product, the product is that the TA clones prepared carry
Body, can carry out TA with T4DNA ligases and PCR primer and be connected.
The endonuclease reaction system of table 1
2.TA connections：As shown in table 2, according to purpose PCR fragment：The mol ratio of TA cloning vectors is 3~7: 1 preparation connection
Reaction system, of short duration centrifugation 3-5 seconds after mixing react mixed liquor 5 minutes in 22 DEG C, and reaction end is rearmounted on ice, after progress
Continuous conversion reaction.
The coupled reaction system of table 2
Note：(1) the TA cloning vectors based on digestion added in linked system are 0.03pmol, when purpose PCR fragment
Length advises purpose PCR fragment in below 2000bp：The mol ratio of TA cloning vectors is 7: 1,22 DEG C and connected 5 minutes；Work as mesh
The length of PCR fragment advise purpose PCR fragment in more than 2000bp：The mol ratio of TA cloning vectors is 3: 1,22 DEG C of connections
30 minutes；
(2) consumption (μ l) of purpose PCR fragment=[660 × A × 0.03 × B (ng)] [1000 × purpose PCR fragment is dense by ÷
Spend (ng/ μ l)], wherein A is the base logarithm (bp) of purpose PCR fragment, and B is PCR fragment：The mol ratio of TA cloning vectors.
(3) bacterial strain of conversion, which should try one's best, selects without laqI genes, such as DH5a, TOP10, T1 etc., need to such as use JM109
Bacterial strain, should add a small amount of IPTG on plating medium.
Embodiment 4
As shown in table 3, according to purpose PCR fragment (about 150bp)：The mol ratio of TA cloning vectors is that 7: 1 preparation connections are anti-
System is answered, the purpose PCR fragment is FaMYB5 genes, and its sequence is as shown in SEQ ID NO.2, of short duration centrifugation 3-5 seconds after mixing,
Mixed liquor is reacted 5 minutes in 22 DEG C, reaction end is rearmounted on ice, take whole products conversion DH5a.Fig. 2 is positive bacteria in LB/
Growing state on Amp plating mediums, random 6 bacterium colonies of picking carry out bacterium colony PCR mirror using M13fwd and M13rev primers
It is fixed, as shown in figure 3, the band of result display amplification is identical with expected size (about 500bp), further sequencing display
Target fragment access is completely correct, this demonstrates the TA recombinant vectors positive rate obtained with the present invention close to 100%.
The coupled reaction system of table 3
Some preferred embodiments of invention have shown and described in described above, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and available for various other combinations, modification
And environment, and can be carried out in invention contemplated scope described herein by the technology or knowledge of above-mentioned teaching or association area
Change., then all should be in the appended power of invention and the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention
In the protection domain that profit is required.
SEQUENCE LISTING
<110>Sichuan Agricultural University
<120>A kind of TA cloning vectors and its construction method and application based on digestion
<130> 2017
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 2721
<212> DNA
<213>It is artificial synthesized
<400> 1
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaagtaaa acgacggcca gtttatattc cccagaacat 180
caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc 240
gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc tttcatcccc 300
gatatgcacc accgggtaaa gttcacggga gactttatct gacagtcagt cgatatcgat 360
atcgatatcg atatcgacag acagtcgtgc actggccagg gggatcacca tccgtcgccc 420
cggcgtgtca ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt 480
ataggtgtaa accttaaact gcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 540
ttccacacat tatacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 600
gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 660
gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 720
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 780
cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 840
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 900
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 960
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 1020
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 1080
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 1140
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 1200
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 1260
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 1320
ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 1380
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 1440
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 1500
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 1560
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 1620
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 1680
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctgg ctccccgtcg 1740
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1800
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1860
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1920
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1980
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 2040
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 2100
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 2160
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 2220
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 2280
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 2340
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 2400
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 2460
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 2520
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 2580
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 2640
aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 2700
gtatcacgag gccctttcgt c 2721
<210> 2
<211> 139
<212> DNA
<213>FaMYB5 genes
<400> 2
tcctcggcaa tagatggtcc ctgattgctg ggaggattcc ggggcgaacg gacaatgaga 60
taaagaacta ctggaacact cacctcagta agaagctgat tagtcagggc atagatccca 120
gaacccacaa gccactcaa 139
Claims (7)
1. a kind of TA cloning vectors based on digestion, it is characterised in that the nucleotides sequence of the TA cloning vectors based on digestion
Row are named as pTA03 carriers as shown in SEQ ID NO.1.
2. a kind of construction method of the TA cloning vectors as claimed in claim 1 based on digestion, it is characterised in that including following
Step：Take under the conditions of 3-5 μ g pTA03 carriers, 37 DEG C and to be digested 3-18 hours with restriction enzyme A hdI, digestion terminates
After add a small amount of EcoRV-HF endonuclease digestions 30 minutes, 65 DEG C keep 20 minutes, digestion products using cleavage reagent box it is direct
Post recovery is crossed, or is reclaimed with DNA gel QIAquick Gel Extraction Kit；- 20 DEG C of preservations of recovery product, the product is TA gram prepared
Grand carrier.
3. a kind of application method of the TA cloning vectors as claimed in claim 1 based on digestion, it is characterised in that including following
Step：
Step 1, TA cloning vector of the preparation based on digestion：Take and use restriction enzyme under the conditions of 3-5 μ g pTA03 carriers, 37 DEG C
AhdI is digested 3-18 hours, and digestion adds a small amount of EcoRV-HF endonuclease digestions 30 minutes after terminating, and 65 DEG C are kept for 20 points
Clock, digestion products are directly crossed post using cleavage reagent box and reclaimed, or are reclaimed with DNA gel QIAquick Gel Extraction Kit；Recovery product -20
DEG C preserve, the product is the TA cloning vectors prepared；
Step 2, the TA cloning vectors and purpose PCR fragment mixed preparing coupled reaction body based on digestion for preparing step 1
System, of short duration centrifugation 3-5 seconds after mixing react mixed liquor 5 minutes in 22 degree, and reaction end is rearmounted on ice, carry out follow-up turn
Change reaction.
4. the application method of the TA cloning vectors according to claim 3 based on digestion, it is characterised in that in step 1
Endonuclease reaction system is specially：μ L, the 10U/ μ L of 10xCutSmart Buffer (NEB) 10 restriction enzyme A hdI 2 μ L, 1
The μ g/ μ l μ L of TA cloning vectors 5 based on digestion, surplus is H2O, using upper volume total amount as 100 μ L.
5. the application method of the TA cloning vectors according to claim 3 based on digestion, it is characterised in that in step 2
Coupled reaction system is specially：μ L, the 400U/ μ L of 10xT4 DNA ligase Buffer (NEB) 1 T4 DNA ligase 0.5
The mol ratio of μ L, the 50ng/ μ L μ L of TA cloning vectors 1, purpose PCR fragment and TA cloning vectors is 3~7：1, surplus is
ddH2O, using upper volume total amount as 100 μ L.
6. the application method of the TA cloning vectors according to claim 5 based on digestion, it is characterised in that in linked system
The TA cloning vectors based on digestion addition be 0.03pmol, when the length of purpose PCR fragment is in below 2000bp, mesh
PCR fragment：The mol ratio of TA cloning vectors is 7：1,22 DEG C connects 5 minutes；When purpose PCR fragment length 2000bp with
When upper, purpose PCR fragment：The mol ratio of TA cloning vectors is 3：1,22 DEG C connects 30 minutes.
7. the application method of the TA cloning vectors according to claim 5 based on digestion, it is characterised in that purpose PCR pieces
Consumption (μ l)=[660 × A × 0.03 × B (ng)] ÷ [1000 × purpose PCR fragment concentration (ng/ μ l)] of section, wherein A is mesh
PCR fragment base logarithm (bp), B is PCR fragment：The mol ratio of TA cloning vectors.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5487993A (en) * | 1990-09-27 | 1996-01-30 | Invitrogen Corporation | Direct cloning of PCR amplified nucleic acids |
| KR20010104589A (en) * | 2000-05-15 | 2001-11-26 | 윤경홍 | PCR T/A cloning vector for cloning PCR product by single enzyme digestion and process for preparing thereof |
| CN1590549A (en) * | 2004-06-10 | 2005-03-09 | 清华大学 | Method of constructing T carrier |
| WO2005021747A1 (en) * | 2003-09-01 | 2005-03-10 | Yokohama City University | Novel vector and utilization of the same |
| CN101503698A (en) * | 2009-03-06 | 2009-08-12 | 西南大学 | Non-false positive T vector and preparation |
| CN101948862A (en) * | 2010-09-13 | 2011-01-19 | 原平皓(天津)生物技术有限公司 | T vector construction method |
-
2017
- 2017-03-13 CN CN201710147060.XA patent/CN107058357A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5487993A (en) * | 1990-09-27 | 1996-01-30 | Invitrogen Corporation | Direct cloning of PCR amplified nucleic acids |
| KR20010104589A (en) * | 2000-05-15 | 2001-11-26 | 윤경홍 | PCR T/A cloning vector for cloning PCR product by single enzyme digestion and process for preparing thereof |
| WO2005021747A1 (en) * | 2003-09-01 | 2005-03-10 | Yokohama City University | Novel vector and utilization of the same |
| CN1590549A (en) * | 2004-06-10 | 2005-03-09 | 清华大学 | Method of constructing T carrier |
| CN101503698A (en) * | 2009-03-06 | 2009-08-12 | 西南大学 | Non-false positive T vector and preparation |
| CN101948862A (en) * | 2010-09-13 | 2011-01-19 | 原平皓(天津)生物技术有限公司 | T vector construction method |
Non-Patent Citations (2)
| Title |
|---|
| MESSING,J: "Cloning vector pUC19,complete sequence", 《GENBANK:M77789.2》 * |
| PHILIPPE BERNARD等: "Positive-selection vectors using the F plasmid ccdB killer gene", 《GENE》 * |
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