CN105483188A - Splicing method of DNA fragment - Google Patents
Splicing method of DNA fragment Download PDFInfo
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Abstract
The invention provides a splicing method of a nucleic acid fragment, which comprises: providing a DNA carrier designed with a homologous arm and a specific restriction enzyme cutting site; providing multiple target DNA fragments which have homologous arms and of which heads and tails in adjacent splicing positions are overlapped with each other; cloning the multiple target DNA fragments to be spliced together into the DNA carrier by using seamless cloning, thus obtaining the carrier containing a longer DNA fragment. The fragment obtained by the method can be spliced with other fragments in the same way, thus continuously splicing more fragments together. In gene engineering, the splicing method is suitable for obtaining a large DNA fragment, especially in synthetic biology.
Description
Technical field
The invention belongs to molecular biology and technical field of bioengineering, relate to a kind of DNA fragmentation in genetically engineered operation and clone and joining method.Particularly, the present invention relates to and a kind of multiple DNA fragmentation head and the tail are spliced to form the method for longer DNA fragmentation and the application of the method.
Background technology
DNA (Deoxyribonucleicacid), also known as thymus nucleic acid, is genetic material important in life entity, is also important object and the instrument of life science and biotechnology research.Along with the development of genetic engineering technique and application, in vitro the operating method of DNA is got more and more, such as to amplification, the cutting of DNA molecular, modify and re-assembly, also referred to as recombinant DNA technology, being one of most important achievement in modern molecular biology development, is also engineered core technology.
In these external DNA operations, have an important link to be connected in carrier by DNA fragmentation, thus biology (bacterium, yeast, cell etc.) can be utilized to increase to DNA, screen, the operation such as expression.DNA fragmentation is connected to the DNA clone of process also referred to as narrow sense of carrier.
Can have plasmid, clay, phage, artificial chromosome etc. as DNA vector, wherein plasmid vector is the molecular cloning vector be most widely used.
Realizing the method that DNA segment is cloned in carrier also has multiple.Such as enzyme cuts the method for connection, namely DNA molecular cutting be connected and can have been come by restriction enzyme and ligase enzyme, namely respectively with corresponding restriction enzyme cutting target dna molecule and vector dna molecule, make its two ends obtain containing the outstanding end (sticky end) of part strand respectively, the end (being obtained by same enzyme or isocaudarner cutting) containing complementary sequence forms a complete DNA chain under the effect of DNA ligase.Also there is the method that TA clones, namely respectively add 3 '-dA base at DNA fragmentation end, respectively add 3 '-dT base at carrier end, the connection of DNA fragmentation and carrier can be realized.Also have the method for attachment based on homologous fragment, namely have identical sequence respectively in fragment and linearizing carrier two ends, connected together by the effect of enzyme.
The method realizing the connection of single DNA fragment and carrier is relatively simple and ripe.In method as above, can complete by a step respectively.Under study for action, especially, in synthetic biology application, often need to use the process multiple fragment being connected together the longer DNA molecular of formation one, usual method, first the method for multiple fragment by PCR can be connected together, then clone is connected in carrier.Also can first one of them fragment be cloned in carrier, after forming new carrier, add new fragment again.But these methods all exist obvious shortcoming, there is certain mutation rate in the sequence that the method for PCR obtains, often needs again to screen correct clone, and PCR method institute operable DNA fragmentation quantity and length all limited.The method successively added, on the one hand by the restriction of restriction enzyme site, along with the increase of fragment, optional restriction enzyme site is fewer and feweri, in addition on the one hand, run into fragment many time, the method needs the long time.
In order to solve the problem of multiple fragment assembly, especially a large amount of DNA fragmentation needs the problem being spliced to form longer DNA molecular.The present invention proposes the method that one " recurrence " is cloned, according to carrier and the fragment of the method design, easily, can walk abreast and recursively by the splicing of multiple DNA fragmentation head and the tail, form large DNA molecular.
Summary of the invention
The object of the present invention is to provide a kind of method multiple DNA fragmentation head and the tail being spliced to form longer DNA fragmentation.
Multiple DNA fragmentation is spliced to form a joining method for longer DNA fragmentation, comprises the following steps:
1) a kind of DNA vector is provided, on described carrier, at least order comprises four different restriction enzyme sites, i.e. restriction enzyme site A, restriction enzyme site B, restriction enzyme site C and restriction enzyme site D, wherein, be provided with the left homology arm of carrier between restriction enzyme site A and restriction enzyme site B, between restriction enzyme site C and restriction enzyme site D, be provided with the right homology arm of carrier;
2) multiple target DNA fragments to be spliced is provided, the target DNA fragments head and the tail of contiguous concatenation position are overlapped, and the stem of first target DNA fragments to be spliced is provided with and step 1) the left arm sequence of the left homology arm homology of carrier of described DNA vector, the afterbody of an end fragment to be spliced is provided with and step 1) the right arm sequence of the right homology arm homology of carrier of described DNA vector;
3) by step 2) described in target DNA fragments multiple to be spliced be cloned into step 1 in the lump through the method for seamless clone) described in DNA vector, in the carrier of acquisition, described multiple target DNA fragments to be spliced according to design sequence be connected.
Step 1) in, the key Design of described DNA vector is left and right homology arm and four restriction enzyme sites, and it is special that other designs there is no, and various conventional carrier therefore can be adopted for vector modification acquisition of setting out.Other restriction enzyme sites and characteristic sequence that conventional carrier comprises can be comprised in this carrier.
Embodiment of the present invention used carrier with pUC57 carrier for set out vector modification obtain.
Further, described restriction enzyme site A, restriction enzyme site B, restriction enzyme site C and restriction enzyme site D are all unique in described DNA vector.
Described restriction enzyme site A, restriction enzyme site B, restriction enzyme site C and restriction enzyme site D can be selected from various conventional restriction enzyme site.In the carrier that embodiment is enumerated, restriction enzyme site A, restriction enzyme site B, restriction enzyme site C and restriction enzyme site D are respectively the restriction enzyme site of restriction enzyme SbfI, BpiI, Esp3I and AsiSI in turn.
Preferably, the length of described carrier left arm homologous sequence and carrier right arm homologous sequence is 15-100bp.
As embodiment is enumerated, step 1) sequence of DNA vector used is SEQIDNO:15.
Preferably, during seamless clone, step 1) described in DNA vector linearized.Preferably, DNA vector described in restriction endonuclease B (corresponding restriction enzyme site B) and restriction endonuclease C (corresponding restriction enzyme site C) linearization for enzyme restriction is adopted.
Preferred step 2) in, the overlapped region of target DNA fragments head and the tail of contiguous concatenation position is 15-100bp, preferred 25-100bp.
For preventing the order of connection chaotic, step 2) in, when target DNA fragments to be spliced is in two or more, the regional sequence that the target DNA fragments head and the tail of each contiguous concatenation position are overlapped is different.
Optionally, step 2) in, at least one target DNA fragments to be spliced adopts the method comprised the following steps to obtain:
A) two ends are provided to be attached with and step 1) target DNA fragments to be spliced of the left arm sequence of carrier left homology arm homology described in the DNA vector that provides and the right arm sequence with described carrier right homology arm homology;
B) step fragment a) is cloned into step 1 through the method for seamless clone) in the DNA vector that provides, obtain the carrier that clone has target DNA fragments to be spliced;
C) by step b) after the vector amplification that obtains, cut through restriction endonuclease A and restriction endonuclease C enzyme, or restriction endonuclease B and restriction endonuclease D enzyme are cut, or restriction endonuclease B and restriction endonuclease C enzyme are cut and obtained described target DNA fragments to be spliced.
Further, step c) in, when described carrier cloning has first target DNA fragments to be spliced, adopt restriction endonuclease A and restriction endonuclease C enzyme to cut; When described carrier cloning has last target DNA fragments to be spliced, restriction endonuclease B and restriction endonuclease D enzyme is adopted to cut; If target DNA fragments number to be spliced is more than two, when described carrier cloning has middle target DNA fragments to be spliced, restriction endonuclease B and restriction endonuclease C enzyme is adopted to cut.
Further, step 3), b) in, be cloned into by DNA fragmentation after on carrier, restriction enzyme site and the characteristic sequence of carrier do not change.
Preferably, step 2) in, each target DNA fragments to be spliced all adopts aforesaid method to obtain.
Step 3) obtain the DNA vector that clone has splicing fragment, the mode that enzyme can be adopted to cut is separated the DNA fragmentation spliced.
Method of the present invention can be recycled, the mode of similar recurrence, by step 3) the DNA fragmentation enzyme that spliced in the carrier that obtains is further used as target DNA fragments to be spliced after cutting, for splicing longer DNA fragmentation, DNA fragmentation once or for several times, thus stitchs together by recurrence like this continuously.
Feature of the present invention is to utilize same set of carrier and corresponding restriction enzyme site, is combined the method for seamless clone, two or more DNA fragmentation with head and the tail overlap can be stitched together and form longer fragment.The more important thing is, the fragment be spliced to form thus can be carried out row with other fragments in the same way again and be connect, thus can continuously by more fragment assembly together.In genetically engineered, the DNA fragmentation especially obtaining large section in synthetic biology is very applicable.
Accompanying drawing explanation
Fig. 1 is maternal carrier (pMatric) structural representation
Fig. 2 is target fragment stepwise schematic views
Fig. 3 is that target fragment carrier is carrier (pMatric-Block) builds schematic diagram
Fig. 4 is two fragment one step splicing schematic diagram
Fig. 5 is three fragment one step splicing schematic diagram
Fig. 6 is four fragment one step splicing schematic diagram
Fig. 7 is four fragment two step splicing schematic diagram
Fig. 8 is that maternal carrier in embodiment (pSM1447) builds example
Embodiment
DNA vector of the present invention, may be used for the clone of DNA fragmentation, and with the addition of the carrier after DNA fragmentation, can absorb more DNA fragmentation in the same way; With the addition of the carrier after DNA fragmentation, also can be cut the DNA fragmentation producing and can be loaded in same carrier by suitable enzyme; Recycling by above-mentioned steps, can stitch together continuously by DNA fragmentation.
Step 1) DNA vector used
As shown in Figure 1, carrier of the present invention except as except the due general-purpose attribute of DNA vector, also has:
A, for the left arm sequence (length is 15-100bp) with DNA fragmentation homology;
B, these homologous sequence two ends are chimeric respectively fixing restriction endonuclease A and restriction endonuclease B recognition site;
C, for the right arm sequence (length is 15-100bp) with DNA fragmentation homology;
C, D, these homologous sequence two ends are chimeric respectively fixing restriction endonuclease C and restriction endonuclease D recognition site;
The splicing of single DNA fragment
During splicing for single DNA fragment, left arm sequence and the right arm sequence of homology can be added at the two ends of target DNA fragments.By restriction endonuclease B and this carrier of restriction endonuclease C linearizing.DNA fragmentation and carrier, by the method for seamless clone, connect between the left arm sequence and right arm sequence of homology.
The splicing of multiple DNA fragmentation
For the splicing of multiple DNA fragmentation, first obtained each DNA fragmentation of head and the tail overlapped (overlapping region length 15-100bp) by suitable method, as shown in Figure 2.Add respectively and the left arm sequence of carrier homology and right arm sequence in the stem of first fragment and the afterbody of last fragment.
Subsequently, as shown in Figure 3, each base slice is cloned in carrier by the method for single DNA fragment assembly.
Then, every two adjacent segment can be stitched together by following method:
As shown in Figure 4,
Discharge the carrier pBlock1 containing first fragment by restriction endonuclease A and restriction endonuclease C, obtain fragment 1;
Discharge the carrier pBlock2 containing second fragment by restriction endonuclease B and restriction endonuclease D, obtain fragment 2;
By restriction endonuclease B and any one same carrier of restriction endonuclease C linearizing (pBlock1, pBlock2, or pMatric), obtain carrier;
Two fragments are connected with carrier, fragment 1 and the end to end carrier pBlock12 of fragment 2 can be obtained
The splicing of three or more adjacent fragment every
Three or more adjacent fragment every can be stitched together by following method:
As shown in Figure 5,
Discharge the carrier pBlock1 containing first fragment by restriction endonuclease A and restriction endonuclease C, obtain fragment 1;
Discharge the carrier pBlock2 containing second fragment by restriction endonuclease B and restriction endonuclease C, obtain fragment 2;
Discharge the carrier pBlock3 containing second fragment by restriction endonuclease B and restriction endonuclease D, obtain fragment 3;
By restriction endonuclease B and any one same carrier of restriction endonuclease C linearizing (pBlock1, pBlock2, pBlock3 or pMatric), obtain carrier;
Three fragments are connected with carrier, the end to end carrier pBlock123 of fragment 1,2,3 can be obtained
Fragment is more than splicing when three
When fragment is more than three,
First fragment is discharged by restriction endonuclease A and restriction endonuclease C;
Last fragment is discharged by restriction endonuclease B and restriction endonuclease D;
All the other fragments are all by discharging by restriction endonuclease B and restriction endonuclease C;
4 fragments as shown in Figure 6 are once spliced; Also more fragment is gone for;
Circulation splicing obtains longer DNA fragmentation
When needing the DNA fragmentation of splicing more, can parallel processing.
As shown in Figure 7, according to above-mentioned steps, fragment 1 and fragment 2 are spliced to form fragment 12;
Meanwhile, fragment 3 and fragment 4 are spliced to form fragment 34;
Fragment 12 and fragment 34 can adopt again same thinking to be spliced to form fragment 1234;
Circulation like this, can be spliced to long DNA fragmentation fast.
Below embodiments of the invention are elaborated: the present embodiment is implemented under premised on technical solution of the present invention; give detailed embodiment and concrete operating process; be intended to illustrate the present invention further, but be not used for limiting invention which is intended to be protected.
The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, such as Sambrook etc., molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1
The structure of maternal carrier pSM1447
The preparation of 1.1SM1447 fragment
According to SM1447 fragment sequence, design oligonucleotides DNA molecular is as follows:
SM1447 fragment is prepared according to the method for gene chemical synthesis, and reaction system is as follows:
Response procedures: 95 DEG C of for2min, (95 DEG C of for20sec, 56 DEG C of for30sec, 72 DEG C of for20sec) x20cycles, 72 DEG C of for5min.
After reaction terminates, draw above-mentioned reaction product 0.5 μ L as template, reaction system is as follows:
Response procedures: 95 DEG C of for2min, (95 DEG C of for30sec, 56 DEG C of for30sec, 72 DEG C of for30sec) x30cycles, 72 DEG C of for5min.
After reaction terminates, detect with 1.5% agarose gel electrophoresis, have obvious band at target location 200bp.Reclaim this band with " SanPrep pillar DNA glue reclaims test kit " that Sangon Biotech (Shanghai) Co., Ltd. produces, obtain SM1447 fragment (SEQIDNO:11).
The preparation of 1.2 carrier segments
Carry out enzyme with HindIII and Esp3I restriction enzyme to pUC57 carrier (SEQIDNO:14) to cut, reaction system is as follows:
Endonuclease reaction condition is: 37 DEG C, 4 hours.
Digestion products 1.5% agarose gel electrophoresis detects, and obtains 2.2kb and 426bp two band.The linearized vector fragment of 2.2kb is reclaimed with " SanPrep pillar DNA glue reclaims test kit " that Sangon Biotech (Shanghai) Co., Ltd. produces.
1.3 seamless clones
2.2kb linearized vector fragment in 200bpSM1447 fragment and 1.2 in above-mentioned 1.1 be connected with " instant is seamless Cloning Kit " that Sangon Biotech (Shanghai) Co., Ltd. produces, reaction system is as follows:
Ligation condition is: 50 DEG C, 1 hour.
1.4 screening positive clone
By the connection product conversion e.colistraindh5α competent cell in above-mentioned 1.3, with containing the enterprising row filter of 50mg/L penbritin LB culture medium flat plate after transforming, on the activated rear coating of competence after conversion and above-mentioned flat board, be inverted overnight incubation in 37 DEG C.
Plasmid is extracted, with M13-(-48) to " SanPrep pillar plasmid DNA is extraction agent box in a small amount " that bacterium liquid after the activated cultivation of dull and stereotyped picking bacterial plaque uses Sangon Biotech (Shanghai) Co., Ltd. to produce
(5'-GAGCGGATAACAATTTCACAC-3 ' (SEQIDNO:12)) primer order-checking.Sequencing result comparison is correct, obtains maternal carrier pSM1447 (SEQIDNO:15).
Embodiment 2
Sub-carrier pSM1447-B1, the structure of pSM1447-B2, pSM1447-B3, pSM1447-B4
2.1 vector linearization
Carry out enzyme with Hind III restriction enzyme to the maternal carrier pSM1447 obtained in above-described embodiment 1 to cut, form linearized vector, reaction system is as follows:
Endonuclease reaction condition is: 37 DEG C, 2 hours.
Endonuclease reaction terminates rear use 1.5% agarose gel electrophoresis detection enzyme and cuts entirely, then with " SanPrep pillar PCR primer purification kit " that Sangon Biotech (Shanghai) Co., Ltd. produces, purifying is carried out to endonuclease reaction system, obtain pSM1447 linearized fragment.
The preparation of 2.2 insertion DNA fragmentations
According to the sequence inserting DNA fragmentation, design oligonucleotides DNA molecular is as follows:
Insert DNA fragmentation to be prepared according to the method for gene chemical synthesis, for B1 fragment, reaction system is as follows:
Response procedures: 95 DEG C of for2min, (95 DEG C of for20sec, 56 DEG C of for30sec, 72 DEG C of for20sec) x20cycles, 72 DEG C of for5min.
After reaction terminates, draw above-mentioned reaction product 0.5 μ L as template, reaction system is as follows:
Response procedures: 95 DEG C of for2min, (95 DEG C of for30sec, 56 DEG C of for30sec, 72 DEG C of for40sec) x30cycles, 72 DEG C of for5min.
After reaction terminates, detect with 1.5% agarose gel electrophoresis, have obvious band at target location 610bp.Reclaim this band with " SanPrep pillar DNA glue reclaims test kit " that Sangon Biotech (Shanghai) Co., Ltd. produces, obtain B1 fragment (SEQIDNO:94).
B2 (SEQIDNO:95), B3 (SEQIDNO:96), the preparation method of B4 (SEQIDNO:97) fragment is with above-mentioned B1, and the size of fragment is respectively 625bp, 556bp, 649bp.
2.3 seamless clones
B1, B2, B3, B4 in pSM1447 linearized fragment and 2.2 in above-mentioned 2.1 are inserted DNA fragmentation and connect respectively by " instant is seamless Cloning Kit " produced with Sangon Biotech (Shanghai) Co., Ltd., and reaction system is as follows:
Ligation condition is: 50 DEG C, 1 hour.
2.4 screening positive clone
By the connection product conversion e.colistraindh5α competent cell in above-mentioned 2.3, with containing the enterprising row filter of 50mg/L penbritin LB culture medium flat plate after transforming, on the activated rear coating of competence after conversion and above-mentioned flat board, be inverted overnight incubation in 37 DEG C.
To dull and stereotyped picking bacterial plaque, and with M13+ (-47)/M13-(-48) primer (M13+:5'-AGGGTTTTCCCAGTCACG-3 ' (SEQIDNO:13), M13-:5'-GAGCGGATAACAATTTCACAC-3'(SEQIDNO:12)) PCR detection is carried out to bacterial plaque, picking bacterial plaque in 15 μ LddH2O, 98 DEG C of sex change 10 minutes.
Reaction system is as follows:
Response procedures: 95 DEG C of for3min, (95 DEG C of for25sec, 56 DEG C of for25sec, 72 DEG C of for1min) x25cycles, 72 DEG C of for5min.
PCR reaction product 1.5% agarose gel electrophoresis detects, the PCR primer that stripe size is correct carries out sequence verification, comparison sequencing result, " SanPrep pillar plasmid DNA is extraction agent box in a small amount " that after the activated cultivation of the bacterial plaque that sequence is correct, bacterium liquid uses Sangon Biotech (Shanghai) Co., Ltd. to produce extracts plasmid, obtain sub-carrier pSM1447-B1 respectively, pSM1447-B2, pSM1447-B3, pSM1447-B4.
Embodiment 3
One step splicing of two fragments
One step splicing of 3.1B1 and B2 fragment
3.1.1 vector linearization
Method is as above-mentioned 2.1.
3.1.2 the preparation of DNA fragmentation is inserted
Sub-carrier pSM1447-B1 restriction enzyme SbfI and Esp3I obtained in above-described embodiment 2 is carried out enzyme cut, sub-carrier pSM1447-B2 restriction enzyme A siSI and BpiI carries out enzyme and cuts, and reaction system is as follows:
Reaction conditions is: 37 DEG C, 4 hours.
After reaction terminates, electrophoresis is carried out with 1.5% sepharose, be separated and obtain the fragment that size is 579bp and 594bp, reclaim this two band with " SanPrep pillar DNA glue reclaims test kit " that Sangon Biotech (Shanghai) Co., Ltd. produces.
3.1.3 seamless clone
B1, B2 in pSM1447 linearized fragment in above-mentioned 3.1.1 and 3.1.2 are inserted DNA fragmentation and connect by " instant is seamless Cloning Kit " produced with Sangon Biotech (Shanghai) Co., Ltd., and reaction system is as follows:
Ligation condition is: 50 DEG C, 1 hour.
3.1.4 screening positive clone
By above-mentioned connection product conversion, the method for screening positive clone is with above-mentioned 2.4.Order-checking comparison is correct, obtains pSM1447-B12 carrier.
One step splicing of 3.2B3 and B4 fragment
3.2.1 vector linearization
Method is as above-mentioned 2.1.
3.2.2 the preparation of DNA fragmentation is inserted
Sub-carrier pSM1447-B3 restriction enzyme SbfI and Esp3I obtained in above-described embodiment 2 is carried out enzyme cut, sub-carrier pSM1447-B4 restriction enzyme A siSI and BpiI carries out enzyme and cuts, and reaction system is as follows:
Reaction conditions is: 37 DEG C, 4 hours.
After reaction terminates, electrophoresis is carried out with 1.5% sepharose, be separated and obtain the fragment that size is 525bp and 618bp, reclaim this two band with " SanPrep pillar DNA glue reclaims test kit " that Sangon Biotech (Shanghai) Co., Ltd. produces.
3.2.3 seamless clone
B3, B4 in pSM1447 linearized fragment in above-mentioned 3.2.1 and 3.2.2 are inserted DNA fragmentation and connect by " instant is seamless Cloning Kit " produced with Sangon Biotech (Shanghai) Co., Ltd., and reaction system is as follows:
Ligation condition is: 50 DEG C, 1 hour.
3.2.4 screening positive clone
By above-mentioned connection product conversion, the method for screening positive clone is with above-mentioned 2.4.Order-checking comparison is correct, obtains pSM1447-B34 carrier.
Embodiment 4
One step splicing of three fragments
One step splicing of 4.1B1, B2, B3 tri-fragments
4.1.1 vector linearization
Method is as above-mentioned 2.1.
4.1.2 the preparation of DNA fragmentation is inserted
Sub-carrier pSM1447-B1 restriction enzyme SbfI and Esp3I obtained in above-described embodiment 2 is carried out enzyme cut, sub-carrier pSM1447-B2 restriction enzyme Esp3I and BpiI carries out enzyme and cuts, sub-carrier pSM1447-B3 restriction enzyme A siSI and BpiI carries out enzyme and cuts, and reaction system is as follows:
Reaction conditions is: 37 DEG C, 4 hours.
After reaction terminates, carry out electrophoresis with 1.5% sepharose, separation obtains size and is respectively 579bp, 563bp, the fragment of 525bp, reclaims this three band with " SanPrep pillar DNA glue reclaims test kit " that Sangon Biotech (Shanghai) Co., Ltd. produces.
4.1.3 seamless clone
B1, B2, B3 in pSM1447 linearized fragment in above-mentioned 4.2.1 and 4.2.2 are inserted DNA fragmentation and connect by " instant is seamless Cloning Kit " produced with Sangon Biotech (Shanghai) Co., Ltd., and reaction system is as follows:
Ligation condition is: 50 DEG C, 1 hour.
4.1.4 screening positive clone
By above-mentioned connection product conversion, the method for screening positive clone is with above-mentioned 2.4.Order-checking comparison is correct, obtains pSM1447-B123 carrier.
One step splicing of 4.2B2, B3, B4 tri-fragments
4.2.1 vector linearization
Method is as above-mentioned 2.1.
4.2.2 the preparation of DNA fragmentation is inserted
Sub-carrier pSM1447-B2 restriction enzyme SbfI and Esp3I obtained in above-described embodiment 2 is carried out enzyme cut, sub-carrier pSM1447-B3 restriction enzyme Esp3I and BpiI carries out enzyme and cuts, sub-carrier pSM1447-B4 restriction enzyme A siSI and BpiI carries out enzyme and cuts, and reaction system is as follows:
Reaction conditions is: 37 DEG C, 4 hours.
After reaction terminates, carry out electrophoresis with 1.5% sepharose, separation obtains size and is respectively 594bp, 494bp, the fragment of 618bp, reclaims this three band with " SanPrep pillar DNA glue reclaims test kit " that Sangon Biotech (Shanghai) Co., Ltd. produces.
4.2.3 seamless clone
B2, B3, B4 in pSM1447 linearized fragment in above-mentioned 4.2.1 and 4.2.2 are inserted DNA fragmentation and connect by " instant is seamless Cloning Kit " produced with Sangon Biotech (Shanghai) Co., Ltd., and reaction system is as follows:
Ligation condition is: 50 DEG C, 1 hour.
4.2.4 screening positive clone
By above-mentioned connection product conversion, the method for screening positive clone is with above-mentioned 2.4.Order-checking comparison is correct, obtains pSM1447-B234 carrier.
Embodiment 5
One step splicing of four fragments
5.1 vector linearization
Method is as above-mentioned 2.1.
The preparation of 5.2 insertion DNA fragmentations
Sub-carrier pSM1447-B1 restriction enzyme SbfI and Esp3I obtained in above-described embodiment 2 is carried out enzyme cut, sub-carrier pSM1447-B2 and pSM1447-B3 restriction enzyme Esp3I and BpiI carries out enzyme and cuts, sub-carrier pSM1447-B4 restriction enzyme A siSI and BpiI carries out enzyme and cuts, and reaction system is as follows:
Reaction conditions is: 37 DEG C, 4 hours.
After reaction terminates, carry out electrophoresis with 1.5% sepharose, separation obtains size and is respectively 579bp, 563bp, the fragment of 495bp, 618bp, reclaims this four band with " SanPrep pillar DNA glue reclaims test kit " that Sangon Biotech (Shanghai) Co., Ltd. produces.
5.3 seamless clones
B1, B2, B3, B4 in pSM1447 linearized fragment and 5.2 in above-mentioned 5.1 are inserted DNA fragmentation and connect by " instant is seamless Cloning Kit " produced with Sangon Biotech (Shanghai) Co., Ltd., and reaction system is as follows:
Ligation condition is: 50 DEG C, 1 hour.
5.4 screening positive clone
By above-mentioned connection product conversion, the method for screening positive clone is with above-mentioned 2.4.Order-checking comparison is correct, obtains pSM1447-B1234 carrier.
Embodiment 6
Two step splicings of four fragments
6.1 vector linearization
Method is as above-mentioned 2.1.
The preparation of 6.2 insertion DNA fragmentations
Carrier pSM1447-B12 restriction enzyme SbfI and Esp3I obtained in above-described embodiment 3 is carried out enzyme cut, carrier pSM1447-B34 restriction enzyme A siSI and BpiI carries out enzyme and cuts, and reaction system is as follows:
Reaction conditions is: 37 DEG C, 4 hours.
After reaction terminates, electrophoresis is carried out with 1.5% sepharose, be separated and obtain the fragment that size is 1121bp and 1091bp, reclaim this two band with " SanPrep pillar DNA glue reclaims test kit " that Sangon Biotech (Shanghai) Co., Ltd. produces.
6.3 seamless clones
B12, B34 in pSM1447 linearized fragment and 6.2 in above-mentioned 6.1 are inserted DNA fragmentation and connect by " instant is seamless Cloning Kit " produced with Sangon Biotech (Shanghai) Co., Ltd., and reaction system is as follows:
Ligation condition is: 50 DEG C, 1 hour.
6.4 screening positive clone
By above-mentioned connection product conversion, the method for screening positive clone is with above-mentioned 2.4.Order-checking comparison is correct, obtains pSM1447-B1234 carrier.
Claims (10)
1. a joining method for DNA fragmentation, comprises the following steps:
1) a kind of DNA vector is provided, on described carrier, at least order comprises four different restriction enzyme sites, i.e. restriction enzyme site A, restriction enzyme site B, restriction enzyme site C and restriction enzyme site D, wherein, be provided with the left homology arm of carrier between restriction enzyme site A and restriction enzyme site B, between restriction enzyme site C and restriction enzyme site D, be provided with the right homology arm of carrier;
2) multiple target DNA fragments to be spliced is provided, the target DNA fragments head and the tail of contiguous concatenation position are overlapped, and the stem of first target DNA fragments to be spliced is provided with and step 1) the left arm sequence of the left homology arm homology of carrier of described DNA vector, the afterbody of an end fragment to be spliced is provided with and step 1) the right arm sequence of the right homology arm homology of carrier of described DNA vector;
3) by step 2) described in target DNA fragments multiple to be spliced be cloned into step 1 in the lump through the method for seamless clone) described in DNA vector, in the carrier obtained, described multiple target DNA fragments to be spliced is connected according to the splicing order of design.
2. the method for claim 1, is characterized in that, the length of described carrier left arm homologous sequence and carrier right arm homologous sequence is 15-100bp.
3. the method for claim 1, is characterized in that, described restriction enzyme site A, restriction enzyme site B, restriction enzyme site C and restriction enzyme site D are respectively the restriction enzyme site of restriction enzyme SbfI, BpiI, Esp3I and AsiSI in turn.
4. the method for claim 1, is characterized in that, step 1) sequence of DNA vector used is SEQIDNO:15.
5. the method for claim 1, is characterized in that, step 2) in, the overlapped region of target DNA fragments head and the tail of contiguous concatenation position is 15-100bp.
6. the method for claim 1, is characterized in that, during seamless clone, step 1) described in being limited property of DNA vector restriction endonuclease B and restriction enzyme C linearization for enzyme restriction.
7. the method for claim 1, is characterized in that, step 2) in, at least one target DNA fragments to be spliced adopts the method comprised the following steps to obtain:
A) two ends are provided to be attached with and step 1) target DNA fragments to be spliced of the left arm sequence of carrier left homology arm homology described in the DNA vector that provides and the right arm sequence with described carrier right homology arm homology;
B) step fragment a) is cloned into step 1 through the method for seamless clone) in the DNA vector that provides, obtain the carrier that clone has target DNA fragments to be spliced;
C) by step b) after the vector amplification that obtains, cut through restriction endonuclease A and restriction endonuclease C enzyme, or restriction endonuclease B and restriction endonuclease D enzyme are cut, or restriction endonuclease B and restriction endonuclease C enzyme are cut and obtained described target DNA fragments to be spliced.
8. method as claimed in claim 7, is characterized in that, step c) in, when described carrier cloning has first target DNA fragments to be spliced, adopt restriction endonuclease A and restriction endonuclease C enzyme to cut; When described carrier cloning has last target DNA fragments to be spliced, restriction endonuclease B and restriction endonuclease D enzyme is adopted to cut; If target DNA fragments number to be spliced is more than two, when described carrier cloning has middle target DNA fragments to be spliced, restriction endonuclease B and restriction endonuclease C enzyme is adopted to cut.
9. the method as described in claim as arbitrary in claim 1-8, is characterized in that, described method also comprises: 4) by step 3) the DNA vector enzyme cutting that obtains is from the DNA fragmentation spliced.
10. method as claimed in claim 9, it is characterized in that, described method also comprises: using step 4) DNA fragmentation that completes of the splicing that obtains as target DNA fragments to be spliced, the DNA fragmentation that splicing is longer further.
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| CN110885853A (en) * | 2018-09-11 | 2020-03-17 | 河南农业大学 | Method for constructing multi-sgRNA expression vector |
| CN111705055A (en) * | 2020-08-19 | 2020-09-25 | 江苏集萃药康生物科技有限公司 | DNA molecular cloning method |
| CN112852849A (en) * | 2019-12-31 | 2021-05-28 | 湖北伯远合成生物科技有限公司 | System and method for seamless assembly of large-fragment DNA |
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| CN101016551A (en) * | 2007-02-01 | 2007-08-15 | 南京师范大学 | Method of introducing a plurality of DNA fragments simultaneously into DNA vector |
| CN103215296A (en) * | 2013-03-22 | 2013-07-24 | 武汉伯远生物科技有限公司 | Multi-fragment DNA molecule assemble method and application |
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| CN101016551A (en) * | 2007-02-01 | 2007-08-15 | 南京师范大学 | Method of introducing a plurality of DNA fragments simultaneously into DNA vector |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110885853A (en) * | 2018-09-11 | 2020-03-17 | 河南农业大学 | Method for constructing multi-sgRNA expression vector |
| CN112852849A (en) * | 2019-12-31 | 2021-05-28 | 湖北伯远合成生物科技有限公司 | System and method for seamless assembly of large-fragment DNA |
| CN112852849B (en) * | 2019-12-31 | 2023-03-14 | 湖北伯远合成生物科技有限公司 | System and method for seamless assembly of large-fragment DNA |
| CN111705055A (en) * | 2020-08-19 | 2020-09-25 | 江苏集萃药康生物科技有限公司 | DNA molecular cloning method |
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