CN103642829A

CN103642829A - High-efficiency gene directional cloning method

Info

Publication number: CN103642829A
Application number: CN201310675252.XA
Authority: CN
Inventors: 辛文; 耿亮
Original assignee: BeiJing TransGen Biotech Co Ltd
Current assignee: Beijing quanshijin Biotechnology Co.,Ltd.
Priority date: 2013-12-13
Filing date: 2013-12-13
Publication date: 2014-03-19
Anticipated expiration: 2033-12-13
Also published as: CN103642829B

Abstract

The invention discloses a high-efficiency gene directional cloning method. The method realizes high-efficiency gene directional cloning by utilizing 5-minute rapid connection of Topoismerase I under the room temperature and an in-vitro cyclizing principle. The method comprises the following steps: designing a carrier with a Topoismerase I enzyme recognition site and a directional sequence, and a directional primer, preparing a PCR (Polymerase Chain Reaction) segment and constructing a recombinant vector of a target gene. The used carrier has all regulating elements of a pronucleus expression carrier, and therefore, the PCR product can be connected with the carrier; the high-efficiency gene directional cloning method can be directly used for pronucleus expression without the need of determining connecting direction of the target gene after PCR identification.

Description

A kind of method of high-level efficiency gene directed cloning

Technical field

The present invention relates to the method for a kind of high efficiency gene directed cloning of bioengineering field.

Background technology

Gene clone technology is an important technology in molecular biology, and experts and scholars have explored a lot of different cloning process both at home and abroad, has non-directional clone and directed cloning, and has set up accordingly variety carrier.Although because non-directional clone's method is easy and simple to handle, can access higher clone's number, follow-up evaluation Comparision is complicated, and comparatively conventional directed cloning comprises 2 kinds of methods at present:

(1) traditional T4DNA ligase enzyme connection method

Restriction enzyme digestion carrier and fragment produce the sticky end of complementary pairing, under the effect of T4DNA ligase enzyme, form recombinant molecule.Its principle is: at the two ends of goal gene fragment, design respectively two restriction enzyme sites (identical with the restriction enzyme site on carrier, guarantee that fragment direction of insertion is correct), the specific restriction enzyme of rear utilization, carries out with T4DNA ligase enzyme, connecting after double digestion to the fragment of carrier and PCR product respectively.The shortcoming of the method is: 1, carrier and fragment comprise restriction enzyme site.Goal gene fragment need to be compared and sequential analysis according to the restriction enzyme site of carrier; guarantee that sequence is inner without selected restriction enzyme site, design primer, adds respectively restriction enzyme site and protection base to the two ends of fragment; again, after PCR, obtain the gene fragment order with restriction enzyme site.2, after enzyme is cut, need to carry out purifying to PCR product, this process can adopt column method or rubber tapping method, consuming time longer, and to a certain extent may damage dna fragment, or cause sudden change.3, T4DNA ligase enzyme is longer action time, generally needs under specific temperature of reaction again, needs to react the connection of spending the night or just can complete fragment for a plurality of hours.4, T4DNA ligase enzyme is connected with the triple collision of fragment and carrier, and probability and successful that its three molecule collides are simultaneously all lower.

(2) DNA topoisomerase enzyme process

Nineteen ninety, American scientist Shuman S. finds vaccinia virus DNA topoisomerase I, within 1991, he proves this enzyme spcificity and identifies CCCTT, and within 2 minutes, can complete 80% ligation, thereby for carrying out rapid molecular clone, application vaccinia virus DNA topoisomerase I provides theoretical basis (Sekiguchi J, Cheng C, Shuman S Kinetic analysis of DNA and RNA strand transfer reactions catalyzed by vaccinia topoisomerase.J Biol Chem.1997Jun20; 272 (25): 15721-8).

1999, the efficiency of Shuman S. proof DNA and DNA topoisomerase I covalent coupling can reach 93% (Cheng C, Shuman S.Site-specific DNA transesterification by vaccinia topoisomerase:role of specific phosphates and nucleosides.Biochemistry.1999Dec14 for 5 minutes in room temperature; 38 (50): 16599-612).Due to the outstanding properties of Topoisomerase, it has been successfully applied to TA clone and directed cloning.

Take Invitrogen company utilizes Topoisomerase I to realize directed cloning as example, its principle of design is that carrier one end is 4 base overhangs, and the other end is flat end, when flush end Insert Fragment is connected with carrier, blunt-ended fragment 5 '-OH attacks overhang, realizes and connecting.Although this method can realize directed cloning, cloning efficiency low (www.invitrogen.com).In addition, Cheng C, report in 2000, utilizing the endogenous double-stranded DNA fracture damage that topoisomerase I causes to repair approach---restructuring flank connection approach realizes directed cloning (Cheng C, Shuman S.Recombinogenic flap ligation pathway for intrinsic repair of topoisomerase IB-induced double-strand breaks.Mol Cell Biol.2000Nov; 20 (21): 8059-68).In this approach, vaccinia virus topoisomerase is covalently bonded in the flat end of DNA, and catalysis its with D N A two strands covalently bound with 5 ' indentation and 3 ' outstanding afterbody.The shortcoming of the method is to attack at random DNA double chain with " flap ", thereby cloning efficiency is low.

Summary of the invention

In view of above-mentioned the deficiencies in the prior art, the technical problem to be solved in the present invention is to provide that lower 5 minutes of a kind of Topoisomerase of utilization I room temperature connects fast and external cyclisation principle realizes the method for high-level efficiency directed cloning.The method advantage: design by specific primer, be connected with specific carrier, can complete directed cloning in 5 minutes, cloning efficiency reaches 100%, and is not subject to the restriction of clone gene sequence.

For solving the problems of the technologies described above, the technical scheme that the present invention takes is:

A kind of method that the invention provides high-level efficiency gene directed cloning, the method comprises the following steps:

(1). carrier is prepared: described carrier one end is the flat end of coupling topoisomerase I, and the other end is directed cloning or the expression vector of the overhang that comprises 6 bases;

(2). design of primers

Article one, primer is non-directional primer, another primer is directed primer, wherein, described conventional primer is without extra base with without phosphorylation and primer Insert Fragment DNA complementation, 5 ' end of described directed primer be followed successively by with step (1) in the base sequence of 6 base complementrities pairings of overhang on carrier and the complementary sequence " AAGGG " of topoisomerase I recognition site, and phosphorylation;

(3) pcr amplification, obtains Insert Fragment DNA

Use the primer pair template DNA of step (2) to carry out pcr amplification;

(4) reaction

By carrier, Insert Fragment DNA, topoisomerase I mixes, and room temperature 5 minutes obtains cyclisation product.

In this step, three reactions occur simultaneously, (1) do not comprise additional sequences, Insert Fragment one end without phosphate is connected with one end of the carrier of coupling Topoisomerase I, the Insert Fragment one end that comprises extra base and phosphate, due to the existence of its phosphate, cannot be with coupling one end of Topoisomerase I carrier be connected; (2), there is the sticky end that cleavage reaction produces 6 bases in Topoisomerase I identification Insert Fragment Topoisomerase I site.(3) self-cyclisation,, there is cyclisation in the sticky end complementary pairing of the sticky end of 6 bases and carrier.As shown in Figure 2.

Further, in step (1), the full length sequence of carrier is as shown in sequence table SEQ ID NO.1.Further, described topoisomerase I recognition site is " CCCTT ".Further, aforesaid method also comprises: the cyclisation after product that step (4) is obtained proceeds to competent escherichia coli cell, and incubated overnight, then identifies positive recombinant.

The method of identifying positive recombinant comprises:

(1) PCR method is identified positive recombinant

Selected clone is placed in 10 μ l sterilized waters, and vortex mixed is got 1 μ l mixed solution in 25 μ l reaction systems, with directed primer and non-directional primer, identifies recon.

PCR reaction conditions: 94 ℃ of denaturations 10 minutes (lysing cell, inactivation nuclease), 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 30 circulations of 72 ℃ of extensions (determining the extension time according to the size of fragment), extend 10 minutes after 72 ℃.

(2) restricted enzyme cutting analysis positive recombinant

Select positive recombinant, incubated overnight.With test kit, extract in a small amount plasmid, select suitable restriction enzyme, enzyme is cut evaluation recon.

(3) order-checking: with directed primer and the order-checking of non-directional primer, determine the exactness of sequence.The carrier that the present invention builds and the directed cloning technology providing can be widely used in direct directed cloning and the expression of PCR product, there is very high using value, it is advantageous that: 1, PCR product without enzyme cut, purifying, with the method for simple and fast, realize slewing in 5 minutes clone.If 2 the method are applied on expression vector, as in the present embodiment ( -D1cloning Vector), directed cloning PCR product in the carrier of the high efficient expression of T7lac promotor in directly express, simplified control, recombinant vectors can carry out immediately the work that waits of protein expression after identifying.3, the recombinant vectors obtaining through the method, the success ratio of order-checking improves a lot, can guarantee that 100% is accurate.

Accompanying drawing explanation

Fig. 1: carrier

the collection of illustrative plates of-D1cloning Vector

Fig. 2: use the inventive method to realize high-level efficiency directed cloning schematic diagram;

PCR in Fig. 3: embodiment 1 identifies positive colony.Insert respectively 3 object fragment 1kb, 2kb, the PCR qualification result of 3kb.Wherein: swimming lane M:Trans2K Plus II DNA Marker, swimming lane 1-20: insert gene fragment

Embodiment

The invention provides a kind of directed cloning method, for making object of the present invention, technical scheme and effect clearer, clear and definite, below the present invention is described in more detail.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.Material and source used in embodiment are respectively: carrier

-D1cloning Vector (Beijing Quanshijin Biotechnology Co., Ltd), primer synthetic (the handsome Bioisystech Co., Ltd in Beijing), Topoisomerase I (Beijing Quanshijin Biotechnology Co., Ltd).T4polyneuclotide kinase, 10 * NEB DNA Ligase Buffer (New England Biolabs),

fastPfu DNA Polymerase (Beijing Quanshijin Biotechnology Co., Ltd), (Trans1-T1chemically competent cell (Beijing Quanshijin Biotechnology Co., Ltd),

genomic DNA Kit (Beijing Quanshijin Biotechnology Co., Ltd),

plus II DNA Marker (Beijing Quanshijin Biotechnology Co., Ltd)

Embodiment 1:

With

the fragment of 3 different lengthss of FastPfu DNA Polymerase amplification, carries out respectively after directed cloning, by the method validation directed cloning effect of PCR.

1. goal gene design of primers is with synthetic

Take human gene group DNA's nucleotide sequence as template, choose three sections of goal gene, gene fragment size is respectively with 1kb, 2kb, 3kb.With primer5.0 software, design respectively three pairs of primers, will after directed primer phosphorylation, carry out again PCR reaction.Primer is in Table 1.

Table 1PCR amplification the primer, DNA fragmentation size

Directed primer phosphorylation

37 ℃ 1 hour.Get 1ul for PCR (50 μ l reaction system)

Goal gene fragment is inserted in 2.PCR preparation

PCR response procedures is: 94 ℃ of denaturations 2 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds, 2kb/ minute in 72 ℃ of extensions, totally 30 circulations; 72 ℃ 10 minutes; After PCR finishes, get 5 μ l in 1.5% agarose gel electrophoresis detection.

3, linked system: 5 μ l

Mix gently, 25 ℃ of room temperatures, place 5 minutes.

4, transform

To connect product

Proceed to respectively in competent escherichia coli cell (Trans1-T1chemically competent cell) incubated overnight.

5, PCR identifies Insert Fragment

In picking flat board, white clone respectively chooses 20 in 10ul sterilized water, vortex concussion.Get 1 μ l as template, carry out follow-up bacterium colony PCR and identify.

PCR reaction system

94 ℃ of PCR response procedures 10 minutes, 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ of X second (depending on fragment length).72 ℃ 5 minutes.

Claims

1. a method for high-level efficiency gene directed cloning, the method comprises the following steps:

(1) carrier is prepared: described carrier one end is the flat end of coupling topoisomerase I, and the other end is directed cloning or the expression vector of the overhang that comprises 6 bases;

(2) design of primers

Article one, primer is conventional primer, another primer is directed primer, wherein, described conventional primer is without extra base with without phosphorylation and primer Insert Fragment DNA complementation, 5 ' end of described directed primer be followed successively by with step (1) in the base sequence of 6 base complementrities pairings of overhang on carrier and the complementary sequence " AAGGG " of topoisomerase I recognition site, and phosphorylation;

(3) pcr amplification, obtains Insert Fragment DNA

Use the primer pair template DNA of step (2) to carry out pcr amplification;

(4) reaction

2. method according to claim 1, is characterized in that, in step (1), the full length sequence of carrier is as shown in sequence table SEQ ID NO.1.

3. method according to claim 1, is characterized in that, described topoisomerase I recognition site is " CCCTT ".

4. method according to claim 1, is characterized in that, the method also comprises: the cyclisation after product that step (4) is obtained proceeds to competent escherichia coli cell, and incubated overnight, then identifies positive recombinant.