CN108660146B - A kind of Topo1 connection method improving PCR fragment compatibility and efficiency - Google Patents
A kind of Topo1 connection method improving PCR fragment compatibility and efficiency Download PDFInfo
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- CN108660146B CN108660146B CN201810495055.2A CN201810495055A CN108660146B CN 108660146 B CN108660146 B CN 108660146B CN 201810495055 A CN201810495055 A CN 201810495055A CN 108660146 B CN108660146 B CN 108660146B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- 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
Abstract
The present invention discloses a kind of Topo1 connection method for improving PCR fragment compatibility and efficiency, and connection transformation system, 20-30 DEG C of reaction 4-6min is added in the pcr amplification product of the no phosphorylation in 5 ' ends or modification；The connection transformation system includes the flat ending factor.The Topo1 connection method provided by the invention for improving PCR fragment compatibility and efficiency, no matter PCR product, which is TA or flat end, to use a linked system to be tested, and improve the efficiency of Topo TA connection, time and the cost for greatly solving scientific research person can be applied to molecular biology and generation sequencing.
Description
Technical field
The present invention relates to a kind of molecular biology fields, and in particular to a kind of PCR fragment compatibility and efficiency of improving
Topo1 connection method.
Background technique
With increasingly developed, the traditional restricted digestion of molecular biology and molecular diagnosis, T4 connection produces PCR
Object it is more demanding, connection the reaction time it is long, positive rate is low, connect certainly rate height, virtually can exponentially increase scientific research person preciousness
Time and cost.In order to make up this defect, a kind of technology connecting DNA fragmentation using vaccinia virus DNA topoisomerase 1 is
TOPO clone technology develops therewith.
As long as the source of PCR fragment includes two kinds, first is that the DNA fragmentation of Taq enzyme amplification, due to extra one of its 3 ' end
A base, the method that TA connection can only be used；Another is exactly the flat end expanded with high fidelity enzyme, using flat end
Connection.Currently, in the market, there are two types of simple Topo connection kits, and one is the DNA fragmentations for the amplification of Taq enzyme above
Topo TA kit, another is specific to the Topo blunt connection kit of blunt；Two kinds of kits cannot
It is general, meanwhile, our experimental data discovery, since structure of the Topo1 to the end dsDNA has very high selectivity,
It prepares in the kit of TA, due to the structure of end DNA, the efficiency of Topo TA connection kit is very low, is former
The 20% of this joint efficiency.
Due to each scientific research person's demand disunity, source, that is, pcr amplification product source of Insert Fragment has diversity, leads
Cause the scientific research person having that Taq enzyme must be used to carry out PCR amplification, and some scientific research persons must use high fidelity enzyme to carry out PCR expansion
Increase, but common TOPO Cloning Kit in the market, such as pEASY-TA ZeroCloning Kit (being only applicable to TA)
PEASY-Blunt ZeroCloning Kit (is only applicable to flat end), itself all has limitation, can not accomplish to be compatible with simultaneously
TA peace end clone.In many cases, the sample of scientific researcher is more precious when being unaware that TA or flat end, can only
It attempts one by one, this considerably increases experiment risk, time and the costs of scientific research person.
Summary of the invention
The object of the present invention is to provide it is a kind of improve PCR fragment compatibility and efficiency Topo1 connection method, no matter PCR
Product is that TA or flat end can use a linked system to be tested, and improve the efficiency of Topo TA connection, pole
The big time for solving scientific research person and cost can be applied to molecular biology and generation sequencing.
The technical scheme adopted by the invention is as follows: a kind of Topo1 connection method improving PCR fragment compatibility and efficiency, it will
Connection transformation system, 20-30 DEG C of reaction 4-6min is added in the no phosphorylation in 5 ' ends or the pcr amplification product of modification；The connection
Transformation system includes the flat ending factor.
Further, the connection transformation system in connection method of the present invention, including 48-52mM Tris.Hcl,
The flat ending that the glycerol and percent by volume that the TOPO1 enzyme of 48-52 μ g/ml, percent by volume are 48-52% are 4-6% because
Son.It is TOPO2.0TA/Blunt-Zero Cloning that applicant, which will more specifically connect transformation system and carry out product number,
Mix。
Pcr amplification product of the invention is not particularly limited, can according to circumstances select using Taq enzyme or high fidelity enzyme into
The product of row PCR amplification, but when requiring PCR amplification the end of primer 5 ' can not phosphorylation or modification, and preferred PCR extends
Time is enough, to ensure the integrality of pcr amplification product；It, can be with the yield and matter of electrophoresis detection product after PCR amplification
Amount, if product only has purpose band, no specific band and primer dimer, can directly be reacted, and otherwise suggest carrying out glue
Recovery purifying.It is then preferably purified if it is using plasmid as the PCR product of template, because template plasmid may also grow bacterium
It falls.
The present invention also provides a kind of connection transformation systems, including the flat ending factor.
Further, the present invention provide it is a kind of more specifically connect transformation system, including 48-52mM Tris.Hcl,
The flat ending that the glycerol and percent by volume that the TOPO1 enzyme of 48-52 μ g/ml, percent by volume are 48-52% are 4-6% because
Son.It is TOPO2.0TA/Blunt-Zero Cloning that applicant, which will more specifically connect transformation system and carry out product number,
Mix。
The flat ending factor of the present invention refers to the single-stranded 5 prime excision enzyme activity with 3 ' -5 ', can expand Taq enzyme
DNA fragmentation end A base is removed, and the enzyme of flat end DNA is formed, such as exonuclease T (Exo T).
The present invention also provides the kits comprising the connection transformation system.
The present invention also provides the connection transformation systems or kit in the application of molecular biological arts, more specifically exists
Application in DNA connection.
Advantage of the present invention referring now to the prior art:
(1) improve the compatibility of PCR fragment: the present invention utilizes the flat ending factor by being added, such as exonuclease T,
The enzyme has 3 ' -5 ' single-stranded 5 prime excision enzyme activity, the DNA fragmentation end A base that Taq enzyme expands can be removed, and forms flat end
DNA is held, and then is connect with blunt vector in Topo Cloning Kit, solves the connection compatibility issue of PCR fragment；
(2) the method for the invention expands to obtain PCR expansion by that can improve cloning efficiency, especially Taq enzyme with conspicuousness
Increase production the joint efficiency of object.
Detailed description of the invention
Fig. 1 is the principle of the present invention figure；
Fig. 2 is 1 bacterium colony of embodiment/bacterium solution PCR qualification result figure；
Fig. 3 is 2 bacterium colonies of embodiment/bacterium solution PCR qualification result figure.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.
Embodiment 1
A kind of TOPO1 connection method improving PCR fragment compatibility, includes the following steps:
1, the no phosphorylation in 5 ' ends or the pcr amplification product of modification can use Taq according to the selection of oneself situation by scientific research person
Enzyme or high fidelity enzyme carry out the product of PCR amplification, the end of primer 5 ' of PCR amplification can not phosphorylation or modification, and extend as far as possible
Time is enough, to ensure the integrality of pcr amplification product；After amplification, the yield and quality of electrophoresis detection product, if product
Only purpose band, no specific band and primer dimer, can directly be reacted, and otherwise suggest carrying out glue recovery purifying.
It is then preferably purified if it is using plasmid as the PCR product of template, because template plasmid may also grow bacterium colony.
2, connection transformation system is prepared: including 2 μ l in the TOPO2.0TA/Blunt-Zero Cloning Mix of 20 μ l
500mM Tris.Hcl (PH7.5), 1 μ g vaccinia virus TOPO1 enzyme, 5 μ gBSA, 50% glycerol, 1 μ l exonuclease T.
3,1 μ l connection transformation system is added in 125ng pcr amplification product, uses H2O supplies volume to 5 μ l, flicks mixing,
Of short duration centrifugation reacts at room temperature 5min.If TOPO2.0 TA/ is added in the amplified production of the Taq series of Taq enzyme such as Vazyme
In Blunt-Zero Cloning Mix, 5min is reacted at room temperature, the flat ending factor in Mix is by Taq enzyme amplified production end
A remove, the phosphodiester bond between flat 5 '-OH attack TOPO of ending product and carrier and TOPO enzyme, final TOPO enzyme quilt
It releases, carrier and flat ending product form annular recon；If the Phanta series of high fidelity enzyme such as Vazyme
Amplified production is added in TOPO 2.0TA/Blunt-Zero Cloning Mix, reacts at room temperature 5min, because itself being flat
Ending product, therefore the phosphodiester bond between 5 '-OH attack TOPO and carrier and TOPO enzyme, final TOPO enzyme are released
Come, carrier and flat ending product form annular recon, the TOPO product of concrete principle Vazyme company as shown in Figure 1:
TOPO2.0TA/Blunt-Zero Cloning Mix can be compatible with band A simultaneously and expand Insert Fragment and flat end expansion Insert Fragment.
And the band A Insert Fragment that is only used for connection Taq enzyme amplification of the competing product for TA connection；Competing product are for blunt connection simultaneously
It is only used for the flat end Insert Fragment of high fidelity enzyme amplification.
As control, made comparisons respectively using the product that existing product in the market is connected for TA connection with Blunt.Example
Such as, 1 μ l TA is added with the segment that 125ng high fidelity enzyme expands in the pcr amplification product that 125ng Taq enzyme expands respectively to connect
Mix in, use H2O supplies volume to 5 μ l, flicks mixing, of short duration centrifugation reacts at room temperature 5min.Respectively by 125ng Taq enzyme
The mix that 1 μ l blunt is connected is added with the segment that 125ng high fidelity enzyme expands for the pcr amplification product of amplification, uses H2O supplies body
Product flicks mixing, of short duration centrifugation reacts at room temperature 5min to 5 μ l.
4, competent cell is taken out from -70 DEG C, is immediately placed in and melts on ice, target DNA is added, and (plasmid or connection produce
Object), it flicks tube wall and mixes and (avoid being inhaled with rifle and beat), stand 30min on ice.After 42 DEG C of water-bath heat shock 30sec, it is immediately placed on ice
2min is stood, is sure not to shake centrifuge tube.900 μ l LB or SOC fluid nutrient mediums (without antibiotic) is added into centrifuge tube, mixes
It is even to be placed on 37 DEG C, recovery 1h in 200rpm shaking table.2,500 × g is centrifuged 3min, 900 μ l supernatants is discarded, with remaining culture medium
After thallus is resuspended, it is uniformly coated in the LB solid medium tablets of the antibiotic of benzyl containing ammonia.Plate is just being placed in 37 DEG C of cultures
Case 10min is inverted plate, is incubated overnight after bacterium solution is completely absorbed.
5, bacterial examination identification or sequencing analysis
Bacterium colony/bacterium solution PCR identification: monoclonal to 10 μ l sterile waters are chosen and are mixed as template；It is recommended to use 2 × Taq
Master Mix(Dye Plus),Vazyme#P112.Concrete outcome is shown in Table 1 and Fig. 2.
1 result of table clones number statistics (a)
| Vazyme-topO | Competing product-TA | Competing product-Blunt | |
| Band A Insert Fragment | 4000 | 1000 | 0 |
| Flat end Insert Fragment | 8000 | 0 | 4000 |
According to the result of table 1 and Fig. 2 it is found that 1 result positive rate of embodiment is identified, Insert Fragment size is 1kb, according to table
Method (TOPO product TOPO2.0TA/Blunt-Zero Cloning Mix) of the present invention known to 1 data is being attached
With 4 times that A Insert Fragment efficiency is competing product, when carrying out flat end Insert Fragment, efficiency is 2 times of competing product.Fig. 2 result is into one
Step proves that method (TOPO2.0TA/Blunt-Zero Cloning Mix) of the present invention can be compatible with band A simultaneously and be inserted into piece
The end Duan Yuping Insert Fragment and there is no problem for positive rate.
Embodiment 2
A kind of TOPO1 connection method improving PCR fragment joint efficiency, includes the following steps:
1, the no phosphorylation in 5 ' ends or the pcr amplification product of modification can be selected to use by scientific research person according to oneself situation
Taq enzyme or high fidelity enzyme carry out the product of PCR amplification, the end of primer 5 ' of PCR amplification can not phosphorylation or modification, and prolong as far as possible
Stretch that the time is enough, to ensure the integrality of pcr amplification product；After amplification, the yield and quality of electrophoresis detection product, if producing
Object only has purpose band, no specific band and primer dimer, can directly be reacted, and otherwise suggests that progress glue recycling is pure
Change.It is then preferably purified if it is using plasmid as the PCR product of template, because template plasmid may also grow bacterium colony.
2, connection transformation system is prepared: including 2 μ l in the TOPO2.0TA/Blunt-Zero Cloning Mix of 20 μ l
500mM Tris.Hcl (PH7.5), 1 μ g vaccinia virus TOPO1 enzyme, 5 μ gBSA, 50% glycerol, 1 μ l exonuclease T.
3,1 μ l connection transformation system is added in the pcr amplification product for expanding 125ng Taq enzyme, uses H2O supplies volume to 5 μ
L, flicks mixing, and of short duration centrifugation reacts at room temperature 5min.TOPO2.0TA/ such as is added in the amplified production of the Taq series of Vazyme
In Blunt-Zero Cloning Mix, 5min is reacted at room temperature, the flat ending factor in Mix is by Taq enzymatic amplification product end
A remove, the phosphodiester bond between flat 5 '-OH attack TOPO of ending product and carrier and TOPO enzyme, final TOPO enzyme quilt
It releases, carrier and flat ending product form annular recon；Concrete principle is as shown in Figure 1.
As control, compared with the mix of existing product TA connection in the market.The PCR amplification that 125ng Taq enzyme is expanded
The mix of 1 μ l TA connection is added in product, uses H2O supplies volume to 5 μ l, flicks mixing, of short duration centrifugation reacts at room temperature 5min.
4, competent cell is taken out from -70 DEG C, is immediately placed in and melts on ice, target DNA is added, and (plasmid or connection produce
Object), it flicks tube wall and mixes and (avoid being inhaled with rifle and beat), stand 30min on ice.After 42 DEG C of water-bath heat shock 30sec, it is immediately placed on ice
2min is stood, is sure not to shake centrifuge tube.900 μ l LB or SOC fluid nutrient mediums (without antibiotic) is added into centrifuge tube, mixes
It is even to be placed on 37 DEG C, recovery 1h in 200rpm shaking table.2,500 × g is centrifuged 3min, 900 μ l supernatants is discarded, with remaining culture medium
After thallus is resuspended, it is uniformly coated in the LB solid medium tablets of the antibiotic of benzyl containing ammonia.Plate is just being placed in 37 DEG C of cultures
Case 10min is inverted plate, is incubated overnight after bacterium solution is completely absorbed.
5, bacterial examination identification or sequencing analysis bacterium colony/bacterium solution PCR identification: monoclonal to 10 μ l sterile waters are chosen and are mixed as mould
Plate；It is recommended to use 2 × Taq Master Mix (Dye Plus), Vazyme#P112.Concrete outcome is shown in Table 2 and Fig. 3.
2 result of table clones number statistics (a)
| Vazyme-topO | Competing product-TA | |
| Band A Insert Fragment | 4000 | 1000 |
2 result positive rate of embodiment identification: Insert Fragment size be 1kb, table 2 as the result is shown: method of the present invention
(TOPO product TOPO2.0TA/Blunt-Zero Cloning Mix) is the 4 of competing product being attached with A Insert Fragment efficiency
Times, Fig. 3 as the result is shown: method (TOPO2.0TA/Blunt-Zero Cloning Mix) of the present invention is in joint efficiency height
At 4 times of Yu Jingpin, there is no problem for positive rate.
Claims (7)
1. a kind of Topo1 connection method for improving PCR fragment compatibility and efficiency, which is characterized in that by 5 ' the no phosphorylations in end
Or connection transformation system, 20-30 DEG C of reaction 4-6min is added in the pcr amplification product of modification；The connection transformation system, including
48-52 mMTris HCl, the TOPO1 enzyme of 48-52 μ g/mL, the glycerol and volume basis that percent by volume is 48-52%
Than the flat ending factor for 4-6%；The flat ending factor is exonuclease T.
2. a kind of connection transformation system, which is characterized in that the TOPO1 including 48-52 mMTris HCl, 48-52 μ g/mL
The flat ending factor that the glycerol and percent by volume that enzyme, percent by volume are 48-52% are 4-6%；The flat ending factor
For exonuclease T.
3. connection transformation system as claimed in claim 2 is in the application of molecular biological arts.
4. application of the connection transformation system as claimed in claim 2 in DNA connection.
5. a kind of kit comprising connecting transformation system described in claim 2.
6. kit described in claim 5 is in the application of molecular biological arts.
7. application of the kit described in claim 5 in DNA connection.
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Address after: Qixia District of Nanjing City, Jiangsu province 210038 Branch Road, Hongfeng Technology Park building C1-2 Patentee after: Nanjing novozan Biotechnology Co., Ltd Address before: Qixia District of Nanjing City, Jiangsu province 210038 Branch Road, Hongfeng Technology Park building C1-2 Patentee before: VAZYME BIOTECH (NANJING) Co.,Ltd. |