CN102604982A - Traceless cloning and reorganizing method by means of activity of exonuclease - Google Patents

Abstract

The invention provides a traceless cloning and reorganizing method by means of activity of exonuclease, which includes: adding DNA (deoxyribonucleic acid) sections with the length of 10-30bp identical with that of sequence at two ends of an insertion site of a cloning carrier to two ends of a target gene respectively, then reorganizing with the carrier DNA under the action of the exonuclease, manly during preparing sections of the target gene, adding the DNA sections with the length of 10-30bp identical with the sequence of the two ends of the insertion site of the cloning carrier respectively to the 5' ends of an amplimer. The traceless cloning and reorganizing method has the advantages that (1) enzyme digestion for the target gene or the DNA sections is omitted, enzyme digestion sites inside the target gene or the DNA sections can be ignored, and the target gene or the DNA sections are free of limits by limit incision enzyme identification sites on the carrier; and (2) unnecessary sequence is avoided introducing when the target genes and the DNA sections are connected to the carrier, namely the traceless cloning.

Description

A kind of seamless clone and recombination method that utilizes exonuclease activity

(1) technical field

The present invention relates to a kind of seamless clone and recombination method that utilizes exonuclease activity.

(2) background technology

Classical gene clone and reconstruction experiment are the basis with II type restriction enzyme and T4DNA ligase enzyme.Utilize restriction enzyme to discern specifically and enzyme is cut DNA, with the T4DNA ligase enzyme different dna fragmentations are linked together then, thereby reach the purpose of gene clone and reorganization.This method exists some problems: at first, all there is proper restriction site in not all gene, and especially the cloning site of some carrier has only under the alternative situation of a few restriction endonuclease; Secondly; After restriction enzyme enzyme clone or reorganization; Can stay unnecessary restriction enzyme enzyme sequence vestige; In most cases the recognition sequence of these restriction enzymes also can cause having more on our target protein some aminoacid sequences subsequently, and these unnecessary amino acid may change the structure and the activity of target protein, and then influence experimental result.

(3) summary of the invention

The object of the invention provides a kind of clone of restriction enzyme site and ligase enzyme and method of recombinant DNA of not relying on.

The technical scheme that the present invention adopts is:

A kind of seamless clone and recombination method that utilizes exonuclease activity; Said method is to add respectively at the goal gene two ends that are inserted into and recombinate under the exonuclease effect with carrier DNA after cloning vector inserts the dna fragmentation that two terminal sequences identical length in site is 10～30bp (being advisable about 15bp); Promptly in preparation during target gene fragment, add respectively that at 5 ' end of amplimer inserting the identical length of site two terminal sequences with cloning vector is the dna fragmentation of 10～30bp (being advisable about 15bp).

Said method comprises:

(1) carrier DNA preparation: insert the corresponding restriction enzyme in site with goal gene and the cloning vector plasmids of purifying is carried out enzyme cut; Perhaps the cloning vector plasmids with dilution is a template, and the sequence of inserting the two ends, site with goal gene designs primer for binding site, and upstream primer is designated as primer1 (about 15bp), and downstream primer is designated as primer2 (about 15bp), carries out pcr amplification;

(2) goal gene preparation: hold the reverse complementary sequence that adds primer1 and primer2 respectively at 5 ' of the original amplimer of goal gene, the gained primer is that template is carried out pcr amplification with the DNA that contains goal gene, obtains goal gene;

(3) recombination: step (1) carrier DNA and step (2) goal gene are mixed, add Pfu archaeal dna polymerase, T4DNA polysaccharase or λ Exonuclease, react the acquisition recombinant DNA.

(4) transform: the recombinant DNA of step (3) gained is transformed into competent escherichia coli cell, and screening obtains to contain the escherichia coli cloning of goal gene plasmid.

Preferably, the used polysaccharase of step (3) is the T4DNA polysaccharase.

Concrete, be SGK2 (serum/glucocorticoid regulated kinase 2) gene ORF with said goal gene, said cloning vector is pBlueScript II KS (-), and inserting the site is that EcoR V restriction enzyme site is an example, and said method is following:

(1) carrier DNA preparation: the cloning vector plasmids with dilution is a template, carries out pcr amplification with primer primer1.1 and primer2.1, obtains carrier DNA;

Primer primer1.1 sequence is following: 5 '-ATCGAATTCCTGCAGCC-3 ';

Primer primer2.1 sequence is following: 5 '-AATCAAGCTTATCGATACCG-3 ';

(2) goal gene preparation: with mouse spinal cord cDNA is template, carries out pcr amplification with primer SGK2F and SGK2R, obtains goal gene;

Primer SGK2F sequence is following:

5’-GGCTGCAGGAATTCGATGGCCTCCAGCCCAGTTG-3’

Primer SGK2R sequence is following:

5’-CGGTATCGATAAGCTTGATCAAGAGTCCAAAATGTCATCATC-3’

(3) recombination: step (1) carrier DNA and step (2) goal gene are mixed, add the T4DNA polysaccharase, carry out 2min at 20 ℃, 70 ℃ of 10min make enzyme deactivation then, and 50 ℃ of temperature bath 30min anneal again; Or add the Pfu archaeal dna polymerase, bathe 30min 50 ℃ of temperature and carry out enzyme and cut and anneal; Or add λ Exonuclease, and carrying out 5min at 37 ℃, 70 ℃ of 10min make enzyme deactivation then, bathe 30min 50 ℃ of temperature again and anneal; Promptly get recombinant DNA.

(4) transform: the recombinant DNA of step (3) gained is transformed into competent escherichia coli cell, and screening obtains to contain the escherichia coli cloning of goal gene plasmid.

Principle of the invention synoptic diagram is seen Fig. 1.Enzymes such as T4, Pfu archaeal dna polymerase have 3 ' → 5 ' exonuclease activity, and (3 ' → 5 ' Exonuclease activity, Figure 1A), and λ Exonuclease (Lambda exonuclease) has 5 ' → 3 ' exonuclease activity (Figure 1B).Under these exonuclease activity effects; The end of double-stranded DNA can form the strand district;, the end between different dna fragmentations (representes with redness and blueness respectively among Fig. 1) when having identical sequence; Just can anneal according to base complementrity paired principle specificity in strand zone, be transformed into the purpose that just can reach seamless reorganization behind the bacterium.When this cloning process requires design PCR primer, 5 ' end of primer to increase respectively with carrier on the identical section of DNA base (SGK2F and SGK2R among Fig. 1 C) of cloning site two terminal sequences.Carrier can be cut (like EcoR V) with enzyme and obtain, and also can use the PCR method amplification vector to obtain in addition.

Beneficial effect of the present invention is mainly reflected in: (1) goal gene or dna fragmentation do not need enzyme to cut, and needn't consider the restriction enzyme site that it is inner, do not receive the restriction of carrier limit property restriction endonuclease recognition site yet; Avoided introducing unnecessary sequence when (2) the purpose fragment is connected to carrier, promptly seamless clone.

(4) description of drawings

Fig. 1 is principle and the design of primers synoptic diagram that utilizes the seamless cloning process of exonuclease activity;

Fig. 2 is the 5 prime excision enzyme activity of Pfu polymerase, T4DNA polymerase and λ Exonuclease; M, 500bp Marker; C, Control, not enzyme-added; The temperature of reaction of Pfu DNA polymerase is 50 ℃, and the temperature of reaction of T4DNApolymerase and λ Exonuclease is 37 ℃.

Fig. 3 is for inserting the fragment detected result; M is marker; Swimming lane 1～8 is that the hickie of 8 random chooses detects the insertion fragment with PCR; Select the bacterial clone of swimming lane 2～4 and 6～8 representatives and do the order-checking evaluation; Wherein the bacterial clone of swimming lane 2,4,6 and 8 representatives all comprises the complete ORF of goal gene SGK2; The clone of swimming lane 3 has then comprised the different transcripts (variant transcript) of SGK2 gene, has only the clone of swimming lane 7 to be non-target gene fragment.

(5) embodiment

Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1: utilize the seamless clone of exonuclease activity SGK (serum/glucocorticoid regulated kinase 2) gene the coding region (open reading frame, ORF) 7.1, detect the 5 prime excision enzyme activity of Pfu, T4DNA polysaccharase and λ Exonuclease

In 10 μ l reaction systems, use Pfu archaeal dna polymerase, T4DNA polysaccharase and the λ Exonuclease (table 1) of 1U to react 5min respectively the 100ng dna fragmentation, behind 10min and the 30min, add 2 μ l 50mM EDTA termination reactions, carry out electrophoresis.

Table 1: exonuclease activity detects

^*：20mM?Tris(pH?8.8)，10mM?KCl，10mM(NH ₄) ₂SO ₄，20mM?MgSO ₄，0.1％TritonX-100；

^**：67mM?Tris(pH?8.8)，17mM(NH ₄) ₂SO ₄，6.7mM?MgCl ₂，1mM?DTT；

^***：67mM?Glycine-KOH(pH?9.5)，2.5mM?MgCl ₂，50μg/ml?BSA，0.2mM?dNTPs。

The result sees Fig. 2, and is very strong by the exonuclease activity of the visible T4DNA polysaccharase of figure, almost all DNA are degraded fully within the 5min, the exonuclease activity of Pfu archaeal dna polymerase a little less than, and the activity of λ Exonuclease falls between.Prompting at the Pfu archaeal dna polymerase at the 50 ℃ of reaction half a hour of excessive dna digestion not; The reaction times of T4DNA polysaccharase can not be oversize, the method that need take to reduce the reaction times and reduce temperature of reaction; The reaction times of λ Exonuclease is controlled at 5min and gets final product.

7.2, the preparation of carrier DNA.

EcoR V restriction enzyme site (GATATC) target gene fragment is cloned into pBlueScript II KS (-) is an example; The available two kinds of methods of the segmental preparation of carrier DNA, respectively as follows: (a) with pBlueScript II KS (-) plasmid of purifying with restriction enzyme EcoR V complete degestion.(b) pBlueScript II KS (-) plasmid with dilution is template (＜1ng is good), is the binding site designed primer with the sequence at two ends, EcoR V site, pcr amplification.Wherein primer corresponds respectively to the zone of the redness on the carrier and blue markings among Fig. 1, and primer title and sequence are following: primer1.1:ATCGAATTCCTGCAGCC; Primer2.1:ATCAAGCTTATCGATACCG.(annotate, primer such in the practical application can be selected in the EcoRV site)

Above-mentioned enzyme cut product through heat treated 20min more than 65 ℃ after, promptly can be used for seamless clone.In addition, utilize phenol/chloroform extracting, ethanol sedimentation, PCR product purification test kit purifying is cut after methods such as glue recovery cut product or PCR product purification with above-mentioned enzyme, promptly can be used for seamless clone.

7.3, the preparation of goal gene dna fragmentation.

With the primer shown in Fig. 1 C:

Primer SGK2F sequence is following:

5 '- GGCTGCAGGAATTCGATGGCCTCCAGCCCAGTTG-3 ' (underscore is the dna sequence dna for adding partly)

Primer SGK2R sequence is following:

5 '- CGGTATCGATAAGCTTGATCAAGAGTCCAAAATGTCATCATC-3 ' (underscore is the dna sequence dna for adding partly)

CDNA does template with mouse spinal cord, pcr amplification SGK2 gene ORF.The product of amplification is with PCR product purification test kit purifying.

7.4, seamless clone.

After DNA (the seeing 7.3) mixing with the carrier DNA (seeing 7.2) of 50ng preparation and 50ng SGK2 gene ORF, be seamless clone with Pfu archaeal dna polymerase, T4DNA polysaccharase and λ Exonuclease respectively.Wherein the enzyme of Pfu archaeal dna polymerase is cut and is annealed at 50 ℃, carries out simultaneously in the temperature bath process of 30min; Being reflected at 20 ℃ and carrying out 2min of T4DNA polysaccharase, 70 ℃ of 10min make enzyme deactivation then, and 50 ℃ of temperature are bathed 30min and are annealed again; λ Exonuclease is reflected at 37 ℃ and carries out 5min, and 70 ℃ then, 10min makes enzyme deactivation, and 50 ℃ of temperature are bathed 30min and annealed again.Product after the annealing is transformed into competent escherichia coli cell, carries out blue hickie screening.

Table 2: the seamless cloning efficiency of different enzymes

The result shows the seamless cloning efficiency the highest (seeing table 2) with the T4DNA polysaccharase.Random choose hickie be PCR and detect whether to contain the insertion fragment, be example (Fig. 3) with the result of T4DNA polysaccharase, the result shows can utilize this method success clone gene.Sequencing result also shows the correct clone that also can both obtain goal gene with Pfu archaeal dna polymerase and λ Exonuclease.

SEQUENCE?LISTING

 

< 110>Hangzhou Pedagogic University

 

< 120>a kind of seamless clone and recombination method that utilizes exonuclease activity

 

<130>

 

<160> 4

 

<170> PatentIn?version?3.4

 

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<212> DNA

<213> Unknown

 

<220>

< 223>artificial sequence

 

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aatcaagctt?atcgataccg 20

 

 

<210> 3

<211> 34

<212> DNA

<213> Unknown

 

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< 223>artificial sequence

 

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< 223>artificial sequence

 

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Claims (4)

1. a seamless clone and recombination method that utilizes exonuclease activity; It is characterized in that said method is to add respectively at the goal gene two ends and recombinate under the exonuclease activity effect with carrier DNA after cloning vector inserts the dna fragmentation that two terminal sequences identical length in site is 10～30bp; Promptly in preparation during target gene fragment, add respectively that at 5 ' end of amplimer inserting the identical length of site two terminal sequences with cloning vector is the dna fragmentation of 10～30bp.

2. the method for claim 1 is characterized in that said method comprises:

(1) carrier DNA preparation: insert the corresponding restriction enzyme in site with goal gene and the cloning vector plasmids of purifying is carried out enzyme cut; Perhaps the cloning vector plasmids with dilution is a template, and the sequence of inserting the two ends, site with goal gene designs primer for binding site, and upstream primer is designated as primer1, and downstream primer is designated as primer2, carries out pcr amplification;

(2) goal gene preparation: hold the reverse complementary sequence that adds primer1 and primer2 respectively at 5 ' of the original amplimer of goal gene, the gained primer is that template is carried out pcr amplification with the DNA that contains goal gene, obtains goal gene;

(3) recombination: step (1) carrier DNA and step (2) goal gene are mixed, add Pfu archaeal dna polymerase, T4DNA polysaccharase or λ Exonuclease, react the acquisition recombinant DNA.

(4) transform: the recombinant DNA of step (3) gained is transformed into after the competent escherichia coli cell, can screens the escherichia coli cloning that obtains to contain the goal gene plasmid.

3. method as claimed in claim 2 is characterized in that the used enzyme of step (3) is the T4DNA polysaccharase.

4. method as claimed in claim 2; It is characterized in that said goal gene is SGK2 (serum/glucocorticoid regulated kinase a 2) gene ORF; Said cloning vector is pBlueScript II KS (-), and inserting the site is EcoR V restriction enzyme site, and said method is following:

(1) carrier DNA preparation: the cloning vector plasmids with dilution is a template, carries out pcr amplification with primer primer1.1 and primer2.1, obtains carrier DNA;

Primer primer1.1 sequence is following: 5 '-ATCGAATTCCTGCAGCC-3 ';

Primer primer2.1 sequence is following: 5 '-AATCAAGCTTATCGATACCG-3 ';

(2) goal gene preparation: with mouse spinal cord cDNA is template, carries out pcr amplification with primer SGK2F and SGK2R, obtains goal gene;

Primer SGK2F sequence is following:

5’-GGCTGCAGGAATTCGATGGCCTCCAGCCCAGTTG-3’

Primer SGK2R sequence is following:

5’-CGGTATCGATAAGCTTGATCAAGAGTCCAAAATGTCATCATC-3’

(3) recombination: step (1) carrier DNA and step (2) goal gene are mixed, add the T4DNA polysaccharase, carry out 2min at 20 ℃, 70 ℃ of 10min make enzyme deactivation then, and 50 ℃ of temperature bath 30min anneal again; Or add the Pfu archaeal dna polymerase, bathe 30min 50 ℃ of temperature and carry out enzyme and cut and anneal; Or add λ Exonuclease, and carrying out 5min at 37 ℃, 70 ℃ of 10min make enzyme deactivation then, bathe 30min 50 ℃ of temperature again and anneal; Promptly get recombinant DNA;

(4) transform: the recombinant DNA of step (3) gained is transformed into competent escherichia coli cell, and screening obtains to contain the escherichia coli cloning of goal gene plasmid.

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