CN106367407A - Preparation method and application of phi29 DNA polymerase - Google Patents
Preparation method and application of phi29 DNA polymerase Download PDFInfo
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- CN106367407A CN106367407A CN201610647512.6A CN201610647512A CN106367407A CN 106367407 A CN106367407 A CN 106367407A CN 201610647512 A CN201610647512 A CN 201610647512A CN 106367407 A CN106367407 A CN 106367407A
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- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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Abstract
The invention discloses a preparation method and application of phi29 DNA polymerase and specifically relates to application of the phi29 DNA polymerase in preparation of a template plasmid of DNA Marker. The method comprises the following steps: construction of the pronucleus of the phi29 DNA polymerase; screening of expression strains; purification of phi29 recombinant protein; etc. A medium is inoculated with the strain; after inducible expression, thalli are collected; and the thalli are subjected to breaking and purifying so as to obtain the phi29 DNA polymerase. When the phi29 DNA polymerase is applicable to preparation of plasmid templates, a series of cumbersome steps like cell culture, collection and cracking of bacteria, and separation and purification of plasmid DNAs can be omitted; an initial sample of a nanogram magnitude can be amplified to obtain DNAs of a microgram magnitude; and the method has the advantages of simple steps, conservation of time, high efficiency, low cost, etc.
Description
Technical field
The invention belongs to molecular biology reagents field and in particular to a kind of preparation method of phi29 dna polymerase and its
Application.
Background technology
Dna marker is dna fragment mixture known to one group of molecular size range, unknown in nucleic acid electrophoresis for indicating
The molecular size range of sample, is one of the most frequently used molecular biology reagents of life science.However, preparation dna marker
Need substantial amounts of plasmid template, and prepare that plasmid template is quite time-consuming, current template construct method, wastes time and energy.And answer
With phi29dna polymerase prepare plasmid template can save cell culture, the collection of antibacterial and cracking and plasmid dna point
From with a series of loaded down with trivial details steps such as purification.Can be directly with cell culture fluid, bacterium colony or a small amount of using phi29dna polymerase
Plasmid is template, and amplification clone obtains taking the plasmid of genes of interest, and this method can produce 3～5g/l template in 4 hours, with
The dna marker preparation continuing after an action of the bowels.Research shows that phi29 dna polymerase, phage t7 dna polymerase and Sequenase all may be used
For plasmid amplification, amplification efficiency in this system for the phi29 dna polymerase is 50ntps；Phage t7dna polymerase
Though there is higher initial synthetic ratio (300ntps) with Sequenase, passage, phage t7dna polymerase and survey in time
Sequence enzymatic synthesiss rate progressively declines, and its final efficiency is than low 10 times about of phi29 dna polymerase.
Phi29 dna polymerase is the mesophilic dna polymerization cloning from bacillus subtilis phage phi29
Enzyme (phi29 dna polymerase), phi29 polymerase tool is high to continue amplification ability (for up to 70kb) and amplification efficiency height,
The interior amplification to target nucleic acid molecule of 30min is up to 1000 times；Meanwhile, there is very strong strand-displacement activity, be suitable for efficient isothermal dna
Amplification.Additionally, phi29 dna polymerase also has 3 ' -5 ' excision enzyme proofreading activities.
But there is presently no the concrete preparation method of open phi29 dna polymerase, more do not disclose high yield
Phi29 dna polymerase is not also applied to the preparation of dna marker template plasmid by the preparation method of phi29 dna polymerase
The record of aspect.
Content of the invention
Therefore, the purpose of the present invention is a kind of preparation method and applications of phi29 dna polymerase, using the method energy
Enough obtain the phi29 dna polymerase of high yield, and can be directly with cell culture fluid, sweet by the phi29 dna polymerase being obtained
Oily bacterium or a small amount of plasmid obtain, for template amplification, the template plasmid preparing dna marker in a large number, have expanded phi29 dna and have gathered
The application of synthase.
On the one hand, the present invention provides a kind of preparation method of phi29 dna polymerase, comprises the following steps:
1) forward primer of gene of design synthesis phi29 dna polymerase and downstream primer, the core of described forward primer
, as shown in seq id no:3, the nucleotide sequence of described downstream primer is as shown in seq id no:4 for nucleotide sequence；
2) pass through pcr amplified reaction, obtain pcr product, and expression vector passes through double digestion, then the pcr after enzyme action is produced
Pcr product is connected after double digestion by thing and expression vector connection again with the expression vector after double digestion, converts escherichia coli,
Sequencing, filters out positive expression recombiant plasmid；
3) by positive expression recombinant plasmid transformed in Host Strains, add derivant inducing culture, to obtain expression strain；
4) finally the expression strain of acquisition is carried out purification, obtain phi29 dna polymerase.
Preferably, in step 2) in, described expression vector carries out double digestion by ndei and xhoi
Preferably, in step 2) in, described expression vector is pet21a expression vector.
Preferably, in step 3) in, described Host Strains are bl21 (de3).
Preferably, in step 3) in, described derivant is iptg.
On the other hand, the present invention also provides a kind of preparation method of above-mentioned phi29 dna polymerase.
Another aspect, the present invention also provides the phi29 dna that a kind of above-mentioned phi29 dna polymerase preparation method is obtained
Application in preparing dna marker template plasmid for the polymerase.
Another further aspect, the present invention also provides a kind of above-mentioned phi29 dna polymerase for preparing the side of dna marker
Method, comprises the following steps:
(1) denaturation contains the Host Strains of amplification purpose plasmid, obtains the Host Strains after denaturation；
(2) using the Host Strains after appropriate denaturation as template, add phi29dna polymerase above-mentioned in right amount and appropriate
Random primer carries out pcr amplification, the purpose plasmid after being expanded；
(3) the purpose plasmid after to expand, as template, by different primer pairs, amplifies different size of respectively
Dna fragment, prepares dna marker.
Preferably, in step (2), after carrying out pcr amplification, also include, by pcr amplification system at 65 DEG C, heating 10
Minute, the step carrying out heat inactivation.
Preferably, in step 3) in, amplify respectively size be 5kb, 3kb, 2kb, 1.5kb, 1kb, 750bp, 500bp,
The dna fragment of 250bp, 100bp.
The present invention has been obtained phi29 dna polymerase, and the conversion microbial activity being obtained by the method is high, yield
High；Additionally, in all known polymerases, phi29 dna polymerase has very high synthesis persistence and (the longest synthesizes
The dna fragment of 70kb length) and strand-displacement activity.
Prepared phi29 dna polymerase can with high yield expand dna, even if micro template also can efficient amplification, can
It is directly used in amplification and obtains substantial amounts of preparation dna marker, and the dna marker steady quality obtaining, band be clear, distribution
Uniformly.In 30min, 1000 times can be reached to the amplification of target nucleic acid molecule.
Phi29 dna polymerase be applied to prepare plasmid template can save cell culture, the collection of antibacterial and cracking with
And a series of loaded down with trivial details steps such as the isolation and purification of plasmid dna.Can also expand from the original samples of nanogram (ng) magnitude
To the dna of Gamma Magnitude, have the advantages that step is simple, save time, efficiently, low cost.
Brief description
Hereinafter, to describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:
Fig. 1 is the result schematic diagram of the expression strain screening high expression in embodiments of the invention 1 by sds electrophoresis,
Wherein, 1: the stock solution after induction, 2: the stock solution not induced, 3: the supernatant of broken bacterium solution, 4: the precipitation of broken bacterium solution, mk:protein
marker；
Fig. 2 is the result carrying out after purification by destination protein in embodiments of the invention 1, wherein, mk:dna marker,
Electrophoresis result under a: reducing condition, b: the electrophoresis result under non-reducing condition；
Fig. 3 is the electrophoresis result overnight expanding puc19 plasmid in embodiments of the invention 2 with not commensurability bacterium solution for template,
Wherein, 1-4 is illustrated respectively in pcr reaction system and adds 0.5 μ l, the electrophoresis result that the template of 1 μ l, 2 μ l, 3 μ l is expanded,
5-8 represents the electrophoresis result that the amplified production in 1-4 carries out ecor i enzyme action, m:1kb dna ladder respectively；
Fig. 4 represents the electrophoresis result of the dna marker preparing in embodiments of the invention 2；
Fig. 5 is the electrophoresis result overnight expanding ppic9k plasmid in embodiments of the invention 3 with not commensurability bacterium solution for template,
Wherein, 1-2 is illustrated respectively in pcr reaction system and adds 1 μ l, the electrophoresis result that the template of 0.5 μ l is expanded, m:1kb
dna ladder；
Fig. 6 represents the electrophoresis result of the dna marker preparing in embodiments of the invention 3.
Specific embodiment
Unless specifically stated otherwise, reagent used in following examples all can from regular distributor available from.
The preparation of embodiment 1.phi29dna polymerase
The structure of 1.phi29 expression plasmid
As shown in seq id no:2, first full genome synthesis phi29dna is polymerized the aminoacid sequence of phi29dna polymerase
The gene order of enzyme, i.e. phi29 gene (seq id no:1), using synthesis phi29f (seq id no:3:
Cgcatatgatgaagcacatgccgcgcaag) primer and phi29r (seqidno:4:
Cgctcgagcttgatggtgaaggtatc) primer carries out pcr amplification, reclaims pcr product, and is connected to ndei and xhoi enzyme
On pet21a expression vector after cutting, convert escherichia coli dh5 α, sequencing obtains correct cloned plasmids.
The expression of 2.phi29 recombiant protein
The correct cloned plasmids obtaining are transformed in Host Strains, the monoclonal thalline after inoculation conversion in test tube.
(1) by monoclonal thalline in 37 DEG C of shaking tables, cultivate 4 hours, add 0.5mm iptg induction phi29 protein expression,
Continue culture 3 hours, collects thalline, rationally number.
(2) all draw a small amount of thalline for each number, add 100 μ l electrophoresis sample-loading buffers, boil 5 minutes, 12 after cooling,
000rpm, is centrifuged 7 minutes, takes supernatant, and polyacrylamide gel electrophoresis detect.
According to the detection of electrophoresis, the clonal expression of No. 4 bacterial strains preferably, therefore selects No. 4 clones to use bacterium as extensive expression
Kind, result is as shown in Figure 1.
The purification of 3.phi29 recombiant protein
(1) take the escherichia coli of the 10g express express target protein of collection, add 100ml balance buffer, use after stirring
Ultrasonic Cell Disruptor fully cracks, 12000rpm, is centrifuged 20min, takes supernatant；And with 0.22 μm of filter filtering and impurity removing；
(2) solution is prepared
1. buffer1:20mm tris, 250mm nacl, 10mm imidazole, ph8.0 are balanced；
Eluting buffer1:20mm tris, 250mm nacl, 500mm imidazole, ph8.0；
2. buffer2:20mm pb, ph7.0 are balanced；
Eluting buffer2:20mm pb, 1m nacl, ph7.0；
3. buffer:pbs, ph 7.4 are preserved.
(3) chromatography purification
1st, Primary purification (ni post)
1. the 0.2m naoh using 5cv (cv: represent the volume of filler in chromatographic column) rinses tomographic system, soaks 3h, inactivation
Endotoxin, goes heat source water to rinse tomographic system to neutrality with 10cv；
2. use the control thermal source 0.2m niso of 5cv4Fully rinse tomographic system, activate chromatography media, remove heat source water with 5cv
Rinse the niso that tomographic system will be dissociated4Rinse well, balance buff 1 with 5cv simultaneously and fully rinse tomographic system；
3. pretreated sample is carried out loading with the flow velocity of 5ml/min, balance buffer 1 with 5cv and fully rinse layer
Analysis system；
4. balance buffer 1 and eluting buffer 1 with 3cv, using step level type of elution (point three steps: 50mm imidazoles is washed
De- liquid, 100mm imidazole elution, 250mm imidazole elution, 500mm imidazole elution) protein of interest；
2nd, moderate purification (strong ion-exchange chromatography)
1. the 0.2m naoh using 5cv rinses tomographic system, soaks 3h, inactivates endotoxin, goes heat source water to rinse layer with 10cv
Analysis system is to neutrality；
2. use the control thermal source 0.2m niso of 5cv4Fully rinse tomographic system, activate chromatography media, remove heat source water with 5cv
Rinse the niso that tomographic system will be dissociated4Rinse well, balance buffer 2 with 5cv simultaneously and fully rinse tomographic system；
3. pretreated sample is carried out loading with the flow velocity of 5ml/min, after completion of the sample, balance buffer with 5cv again
2 abundant flushing tomographic systems；
4. balance buffer2 and eluting buffer2 with 3cv, using one-step elution mode protein of interest；
5. fully rinse tomographic system with the control heat source water of 3cv, tomographic system is rinsed well, then uses 3cv equalizing and buffering
Liquid fully rinses tomographic system；
6. the sample of recovery is carried out loading with the flow velocity of 2ml/min, fully rinse chromatography system with 3cv balance buffer2
System.
(4) post processing
Sample fraction is received according to purification collection of illustrative plates, dialyses to preservation system for 100 times；Take sample after dialysis, -80 DEG C are done freeze thawing
Test, chooses the qualified sample of freeze thawing, makes quality inspection, warehouse-in, and sds polyacrylamide gel electrophoresis detect, result such as Fig. 2 institute
Show: destination protein expression is clear, and stripe size meets, the active 168u/ μ l, yield 1,650,000 u/l of acquisition.
The amplification of embodiment 2.dna marker template plasmid
1. sample denaturation: take the dh5 α bacterium solution containing puc19 plasmid, with reference to following reaction system thermal denaturation；
2. plasmid amplification: carry out by following reaction condition；
3. heat inactivation: 65 DEG C, 10 minutes；
4. it is sequenced: take above-mentioned reactant liquor 2 μ l, add 8 μ l distilled waters, you can direct Sequencing.
Result is as shown in figure 3, from figure 3, it can be seen that directly with bacterium solution as template, micro bacterium solution (0.5 μ l) can expand
Increase and obtain substantial amounts of plasmid template, save time and simple to operate.
5., with the puc19 plasmid of above-mentioned amplification acquisition as template, use pcr method, devise different primer pairs, expand respectively
Increase and the dna fragment that size is 5kb, 3kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp, 100bp, prepare for the first time
Dna marker, then mixes each fragment according to a certain percentage and is optimized.Detect, result is such as through 1% agarose gel electrophoresiies
Fig. 4 can be seen that, prepared dna marker (being named as dl5000) steady quality, band are clear, be evenly distributed.
Embodiment 3, the amplification of dna marker template plasmid
1. sample denaturation: take the frozen dh5 α bacterium solution containing ppic9k plasmid, with reference to following reaction system thermal denaturation；
2. plasmid amplification: carry out by following reaction condition.
3. heat inactivation: 65 DEG C, 10 minutes.
4. it is sequenced: take above-mentioned reactant liquor 2 μ l, add 8 μ l distilled waters, you can direct Sequencing.
Result as shown in figure 5, from fig. 5, it can be seen that directly with glycerol bacterium solution as template, micro glycerol bacterium solution (0.5 μ
L) it is the substantial amounts of plasmid template of amplifiable acquisition, save time and simple to operate.
5. with the ppic9k plasmid of above-mentioned amplification acquisition as template, use pcr method, devise different primer pairs, respectively
Amplify the dna fragment that size is 5kb, 4kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp, prepare for the first time
Dna marker, then mixes each fragment according to a certain percentage and is optimized.Detect, result is such as through 1% agarose gel electrophoresiies
Fig. 6 can be seen that, prepared dna marker (being named as dl5000) steady quality, band are clear, be evenly distributed.
Above-described is only the preferred embodiment of the present invention it is noted that for those of ordinary skill in the art
For, without departing from the concept of the premise of the invention, some deformation can also be made and improve, these broadly fall into the present invention
Protection domain.
Claims (8)
1. a kind of preparation method of phi29dna polymerase, comprises the following steps:
1) forward primer of gene of synthesis phi29dna polymerase and downstream primer, the nucleotide sequence of described forward primer is such as
Shown in seq id no:3, the nucleotide sequence of described downstream primer is as shown in seq id no:4；
2) expanded by pcr, obtain pcr product, then pcr product and expression vector are passed through double digestion, then by the pcr after enzyme action
Product and expression vector are connected with expression vector after double digestion after connecting through double digestion, convert escherichia coli, sequencing, filter out
Positive expression recombiant plasmid；
3) by positive expression recombinant plasmid transformed in Host Strains, add derivant inducing culture, to obtain expression strain；
4) finally the expression strain of acquisition is carried out purification, obtain phi29dna polymerase.
2. the preparation method of phi29dna polymerase according to claim 1 is it is characterised in that in step 2) in, described
Pcr product carries out double digestion by ndei and xhoi.
3. the preparation method of phi29dna polymerase according to claim 1 is it is characterised in that in step 2) in, described
Expression vector is pet21a expression vector.
4. the phi29dna that the preparation method of phi29dna polymerase according to any one of claim 1 to 3 is obtained gathers
Synthase.
5. application in preparing dna marker template plasmid for the phi29dna polymerase according to claim 4.
6. phi29dna polymerase according to claim 4 is used for the method preparing dna marker template plasmid, including
Following steps:
(1) denaturation contains the Host Strains of amplification purpose plasmid, obtains the Host Strains after denaturation；
(2) again using the Host Strains after appropriate denaturation as template, add the phi29dna polymerase described in appropriate claim 4
Carry out pcr amplification with appropriate random primer, the purpose plasmid after being expanded；
(3) the purpose plasmid after to expand, as template, by different primer pairs, amplifies different size of dna piece respectively
Section, prepares dna marker.
7. phi29dna polymerase according to claim 6 be used for prepare dna marker method it is characterised in that
In step (2), after carrying out pcr amplification, also include, by pcr amplification system at 65 DEG C, heating 10 minutes, carrying out heat inactivation
Step.
8. phi29dna polymerase according to claim 6 be used for prepare dna marker method it is characterised in that
Step 3) in, amplify the dna piece that size is 5kb, 3kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp, 100bp respectively
Section.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662893A (en) * | 2020-07-01 | 2020-09-15 | 济南国科医工科技发展有限公司 | Preparation method of molecular diagnostic enzyme preparation |
| CN112805372A (en) * | 2018-10-11 | 2021-05-14 | 深圳华大生命科学研究院 | Phi29 DNA polymerase mutant with improved thermal stability and application thereof in sequencing |
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| CN104328111A (en) * | 2014-10-29 | 2015-02-04 | 北京大学 | Method for high-throughput amplifying plasmid for sequencing |
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2016
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| CN1654650A (en) * | 2004-12-29 | 2005-08-17 | 江苏省血吸虫病防治研究所 | 100bp gradient ribonucleic acid molecular weight marker and its preparation |
| CN104328111A (en) * | 2014-10-29 | 2015-02-04 | 北京大学 | Method for high-throughput amplifying plasmid for sequencing |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112805372A (en) * | 2018-10-11 | 2021-05-14 | 深圳华大生命科学研究院 | Phi29 DNA polymerase mutant with improved thermal stability and application thereof in sequencing |
| CN112805372B (en) * | 2018-10-11 | 2023-10-31 | 深圳华大生命科学研究院 | Phi29 DNA polymerase mutant with improved thermal stability and application thereof in sequencing |
| CN111662893A (en) * | 2020-07-01 | 2020-09-15 | 济南国科医工科技发展有限公司 | Preparation method of molecular diagnostic enzyme preparation |
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