CN112877324B - DNA cloning method - Google Patents

Abstract

The invention discloses a DNA cloning method, which comprises the steps of designing a pair of primers, wherein one end of each primer is matched with a carrier, and the other end of each primer is matched with a target gene fragment; after mixing the target gene segment, the linearized vector and the primer, under the action of exonuclease, the 5 'end bases of the vector segment and the target gene segment are gradually cut off to expose 3' protruding single-stranded region, at this time, the primer can be complementarily paired with the single-stranded region and mutually used as a primer and a template, and is extended under the action of DNA polymerase, so that the vector and the target gene segment have an overlapping region, the extension product is cut into single strands under the action of exonuclease, at this time, the single-stranded regions of the target gene segment and the vector segment can be annealed through complementary pairing, extended under the action of DNA polymerase, and finally connected into circular DNA under the action of ligase. The target gene does not need to be amplified by PCR, so that the mutation caused by PCR is reduced; the target fragment can be specifically cloned from the DNA mixture; no trace of the cleavage site was left.

Description

DNA cloning method

Technical Field

The invention relates to the technical field of biology, in particular to a DNA cloning method.

Background

The classical molecular cloning method relies on a restriction enzyme digestion connection method to clone a gene fragment, the cloned fragment often contains redundant sequences near enzyme digestion sites, and in addition, the larger the gene fragment is, the smaller the enzyme digestion site selectivity is. After the invention of Polymerase Chain Reaction (PCR), the PCR can be widely applied to molecular cloning because the PCR can efficiently amplify target gene fragments. PCR can amplify a target gene fragment by adding a restriction enzyme site and a protective base thereof at the 5' end of a primer, and connecting the target gene fragment into a specific vector after restriction enzyme; or the 5' end of the primer can be added with a sequence matched with the vector to amplify the target gene segment, and the target gene segment can be cloned in an in vitro recombination or in vivo recombination mode.

Despite the diligent efforts of scientists to obtain a number of functionally improved DNA polymerases, PCR amplification still presents several problems: for example, long fragment PCR has high mutation rate and low amplification efficiency. Therefore we need some PCR-dependent small molecular cloning methods as a complement.

The invention application with the publication number of CN102604982A discloses a traceless cloning and recombination method utilizing exonuclease activity, wherein DNA fragments with the same length as the sequences at the two ends of the insertion site of a cloning vector are respectively added at the two ends of a target gene and are 10-30 bp, and then the DNA fragments are recombined with vector DNA under the action of exonuclease, namely, when the target gene fragment is prepared, the DNA fragments with the same length as the sequences at the two ends of the insertion site of the cloning vector are respectively added at the 5' end of an amplification primer. However, in this embodiment, both the vector DNA and the target gene need to be amplified by PCR, and recombinant fragments having the same sequence as that of the insertion site of the cloning vector are added to both ends of the target gene by PCR amplification.

The invention application with the publication number of CN103074358A discloses a simulated recombinant traceless cloning method, which comprises the following steps: (1) the vector DNA preparation: designing a primer by taking a cloning vector plasmid as a template and sequences at two ends of an insertion site of a target gene as binding sites, respectively adding sequences which are matched with the target gene and have the length of 20-30 bp at the 5' end of the primer, marking the obtained upstream primer as primer1, marking the obtained downstream primer as primer2, and carrying out PCR amplification by taking the primer1 and the primer2 as primers to obtain a vector DNA; (2) preparing a target gene: obtaining a segment containing a target gene through enzyme digestion or amplification; (3) gene recombination: mixing the vector DNA in the step (1) with the fragment obtained in the step (2), firstly adding lambda Exonuclease, reacting for 30min at 37 ℃, bathing for 5min at 72 ℃ and 30min at 50 ℃, then cooling to 37 ℃, adding T4 DNA polymerase and dNTPs, and reacting for 15min at 37 ℃ to obtain the recombinant DNA. Similarly, in this embodiment, it is also necessary to use PCR amplification, that is, to use a cloning vector plasmid as a template, to design primers by using sequences at both ends of the insertion site of the target gene as binding sites, and to add sequences of 20 to 30bp in length matching the target gene to the 5' ends of the primers, respectively, to obtain an upstream primer of primer1, a downstream primer of primer2, and to use primer1 and primer2 as primers to perform PCR amplification to obtain vector DNA.

The methods of the invention applications with the publication numbers CN102604982A and CN103074358A need to amplify target gene fragments or vector fragments by PCR. When these fragments have a particularly high or low GC content, are particularly long, have repetitive sequences, have stable secondary structures, etc., PCR tends to be ineffective in amplifying these fragments, and the above-mentioned method cannot be applied. In addition, PCR amplification has the problem of high mutation rate, the longer the fragment is, the higher the mutation rate is, for short fragments, the mutation rate becomes very low by using high-fidelity enzyme amplification, it is easier to screen non-mutated fragments by sequencing, and for long fragments, even if high-fidelity DNA polymerase is used, the mutation rate caused by PCR amplification is increased, and at the same time, the difficulty of screening correct clones by sequencing is multiplied. The present invention is independent of PCR amplification and can avoid the problems of the above methods.

Disclosure of Invention

The present invention provides a PCR-independent DNA cloning method aiming at the above-mentioned disadvantages of the prior art.

A DNA cloning method comprising the steps of:

(1) Providing a double-stranded linearized vector, a double-stranded target gene fragment and a pair of primers, wherein the primers comprise an upstream primer and a downstream primer, two ends of the upstream primer are respectively provided with a pairing region which is complementary to the linearized vector and one end of the target gene fragment in the direction from 3 'to 5', and two ends of the downstream primer are respectively provided with a pairing region which is complementary to the linearized vector and the other end of the target gene fragment in the direction from 3 'to 5';

(2) Under the action of exonuclease, base groups at the 5 'ends of two ends of a double chain of the linearized vector and the target gene fragment are sequentially cut off, a single-stranded region at the 3' end is exposed for complementary pairing with a primer, and the single-stranded region is extended under the action of DNA polymerase after complementary pairing, so that the two ends of the linearized vector and the target gene fragment are provided with pairing regions;

(3) And (3) under the action of exonuclease, sequentially cutting bases at the 5 'ends of the double-stranded vector and the target gene fragment with the pairing regions at the two ends obtained in the step (2), exposing the single-stranded region at the 3' end for complementary pairing between the linearization vector with the pairing region and the target gene fragment, extending under the action of DNA polymerase after complementary pairing, and finally connecting into circular DNA under the action of ligase to obtain a cloning product of cloning the target gene fragment into the linearization vector.

Preferably, the steps (2) and (3) adopt a one-pot reaction, and the double-stranded linearized vector, the double-stranded target gene fragment and the pair of primers, exonuclease, DNA polymerase, ligase and dNTPs in the step (1) are added into a reaction system at the same time.

Wherein the exonuclease is T7 exonuclease; the DNA polymerase is at least one of Taq DNA polymerase, pfu DNA polymerase or KOD DNA polymerase; the ligase is Taq DNA ligase. Of course, other enzymes that meet the requirements can be used in the DNA cloning methods of the present application as well.

When the steps (2) and (3) adopt a one-pot reaction, the reaction system is as follows:

the deficit was made up with ddH 2O. The above-mentioned addition amount is the use concentration or addition amount in the final system, and the general system can be selected from 10. Mu.l system. The Taq DNA ligase buffer can be selected from the existing commonly used buffer system or is commercially purchased and configured.

When the exonuclease is T7 exonuclease, the reaction temperature of the steps (2) and (3) is 45 ℃ and the reaction time is 30min when the one-pot method is adopted.

Preferably, the double-stranded linearized vector in step (1) is obtained by restriction endonuclease cleavage of a circular DNA vector. The double-stranded target gene fragment in the step (1) is obtained by cloning circular DNA of the target gene and performing restriction enzyme digestion.

Preferably, the length of the sequence of the pairing region between the two ends of the upstream primer and the downstream primer in the step (1) and the linearized vector and the target gene fragment is 18bp to 30bp. The length of the sequence of the pairing region is only required to enable the linearized vector, the primer and the target gene fragment to be connected with each other through annealing, so that the length of the sequence is not strictly limited as long as the purpose can be achieved.

And (3) after the cloning product is transformed into an escherichia coli competent cell, screening to obtain the recombinant escherichia coli containing the correct cloning product. Recombinant plasmids containing the target gene fragments can be obtained in large quantities from recombinant Escherichia coli containing the correct cloning products.

Compared with the prior art, the invention has the advantages and effects that:

the target gene is not required to be amplified by relying on PCR (a), the mutation caused by the PCR is reduced, (b) the target fragment can be specifically cloned from a DNA mixture, and (c) no trace of the enzyme cutting site is left.

Drawings

FIG. 1 is a schematic diagram of the principle and process of the primer bridge cloning method.

FIG. 2 is a graph showing the results of detection of T7 exonuclease activity.

FIG. 3 shows an EcoRV cut sequence of Lambda DNA, showing only the terminal bases and the middle bases with-omitted.

FIG. 4 is a graph showing the results of clone identification, and arrows indicate clones with the correct molecular weight.

Detailed Description

The schematic diagram of the "primer bridge cloning method" of the present invention is shown in FIG. 1. A pair of primers (oligonucleotides) is designed, one end of which is paired with the vector and the other end with the target gene fragment. When we mix the target gene fragment, the linearized vector and the pair of primers (fig. 1A), under the action of an enzyme (such as T7 exonuclease) having 5' → 3' exonuclease (5 ' -3 exonuclease) activity, bases at the 5' ends of the vector fragment and the target gene fragment are gradually cut off to expose a 3' overhanging single-stranded region (fig. 1B), at this time, the primers can complementarily pair-anneal with the single-stranded region (Annealing, fig. 1C) and mutually extend under the action of DNA Polymerase (Polymerase) to make the vector and the target gene fragment have an overlapping region (fig. 1D), and the extension product can cut the overlapping region into single strands under the action of 5' → 3' exonuclease (fig. 1E), at this time, the single-stranded regions of the target gene fragment and the vector fragment can be complementarily paired-annealed (fig. 1F), then extended under the action of DNA Polymerase (fig. 1G), and finally ligated under the action of Ligase (Ligase) into a circular DNA for proper cloning screening.

Example 1

1. And detecting the activity of the T7 exonuclease.

T7 exonuclease is double-stranded specificThe 5'→ 3' exonuclease (E) cleaves double-stranded DNA into single-stranded DNA, but does not cleave single-stranded DNA primers. To select appropriate reaction conditions, we first tested the activity of T7 exonuclease. In 10. Mu.l buffer (buffer) (20 mM Tris-HCl,25mM Potasallum acetate,10mM Magnesium acetate,1mM NAD +,10mM DTT,0.1%

X-100, pH 7.6) about 500ng of DL5000DNA Marker was added, and 0.05U of T7 exonuclease was added, and the reaction was carried out at a reaction temperature of 37 ℃ to 50 ℃ for 10 to 90 minutes, respectively.

The results show (FIG. 2) that at 37 ℃ T7 exonuclease completely cleaves all double-stranded DNA within 5kb in length in 10 minutes, indicating that at this temperature T7 exonuclease activity is too high. At 50 ℃ T7 exonuclease was able to cleave completely with 0.1kb DNA fragment in 10 minutes, but fragments above 0.25kb did not change significantly with 10 minutes and 90 minutes, indicating that T7 exonuclease was almost completely inactivated within 10 minutes at 50 ℃. We then examined the activity of T7 exonuclease at 45 ℃ and found that at this temperature T7 exonuclease cleaved the 0.1kb fragment completely in 10 minutes and almost completely at 90 minutes the 0.25kb fragment, indicating that T7 exonuclease was partially inactivated at 45 ℃ for 10 minutes but that a small portion of the residual activity was able to continue to function. At a reaction temperature of about 45 ℃, heat-resistant DNA polymerases such as Taq, pfu and KOD are active, and Taq DNA ligase is also active at this temperature.

2. The efficiency of primer bridging clones was examined.

A1377 bp fragment (shown in SEQ ID No. 1) was cloned from 22 EcoRV fragments of Lambda DNA by the primer bridge cloning method (FIG. 3) to check the efficiency.

2.1, preparing the target DNA fragment.

At 40. Mu.l

In Buffer, 2. Mu.g of LambThe da DNA was reacted with 20U of EcoRV-HF endonuclease at 37 ℃ for 4 hours to cleave the Lambda DNA completely. The endonuclease was then inactivated by heating to 70 ℃ and incubating for 10min, and the final product was Lambda DNA digest at a final concentration of 50 ng/. Mu.l.

TABLE 1 primers used to test the efficiency of primer bridging cloning

Primer Primers	Sequences (5 '-3')
		BP1	ctctagaactagtggatccATCGCATTTTTCACCATGC
BP2	gccccccctcgaggtcgacATCTTTATGTCGAGCAAAGC
		M13F	gtaaaacgacggccag
M13R	caggaaacagctatgac

Note: the upper case letters in the primers indicate the sequence pairing with Labmda DNA and the lower case letters indicate the sequence pairing with the vector.

2.2, preparation of vector DNA fragment.

At 40. Mu.l

In Buffer, 2. Mu.g of pBluescript II KS (-) plasmid (purchased from Stratagene) DNA was reacted with 20U of BamHI-HF and SalI-HF endonucleases at 37 ℃ for 4 hours to prepare pBluescript II KS (-) plasmidThe plasmid DNA was completely digested. Then, the endonuclease was inactivated by heating to 70 ℃ and incubating for 10min, and the resulting product was a pBluescript II KS (-) plasmid DNA cleavage product at a final concentration of 50 ng/. Mu.l.

2.3 primer-bridged cloning.

In about 10. Mu.l of the reaction system, were added:

after mixing uniformly, reacting for 30min at 45 ℃. The reaction product was used for conversion after being placed on ice. After transformation into DH5a competent cells, blue-white screening was performed. The primer BP1 and BP2 sequences are shown in Table 1. Wherein the 1 XTaq DNA ligation Reaction Buffer comprises the following components: 20mM Tris-HCl,25mM Potashium acetate,10mM Magnesium acetate,1mM NAD +,10mM DTT,0.1%

X-100，pH 7.6@25℃。

16 white spots were randomly selected for PCR identification using universal primers M13F and M13R on pBluescript vector. As shown in FIG. 4, 12 of the 16 randomly selected white colonies showed the size of the target gene fragment, and the clones with the size of the target gene fragment contained the correct target gene fragment through sequencing, indicating that the primer-bridge cloning method can effectively clone 1377bp of the target fragment from the mixture of the EcoRV cleavage products of Lambda phage DNA.

Sequence listing

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atcgcatttt tcaccatgct catcaaagac agtaagataa aacattgtaa caaaggaata 60

gtcattccaa ccatctgctc gtaggaatgc cttatttttt tctactgcag gaatataccc 120

gcctctttca ataacactaa actccaacat atagtaaccc ttaattttat taaaataacc 180

gcaatttatt tggcggcaac acaggatctc tcttttaagt tactctctat tacatacgtt 240

ttccatctaa aaattagtag tattgaactt aacggggcat cgtattgtag ttttccatat 300

ttagctttct gcttcctttt ggataaccca ctgttattca tgttgcatgg tgcactgttt 360

ataccaacga tatagtctat taatgcatat atagtatcgc cgaacgatta gctcttcagg 420

cttctgaaga agcgtttcaa gtactaataa gccgatagat agccacggac ttcgtagcca 480

tttttcataa gtgttaactt ccgctcctcg ctcataacag acattcacta cagttatggc 540

ggaaaggtat gcatgctggg tgtggggaag tcgtgaaaga aaagaagtca gctgcgtcgt 600

ttgacatcac tgctatcttc ttactggtta tgcaggtcgt agtgggtggc acacaaagct 660

ttgcactgga ttgcgaggct ttgtgcttct ctggagtgcg acaggtttga tgacaaaaaa 720

ttagcgcaag aagacaaaaa tcaccttgcg ctaatgctct gttacaggtc actaatacca 780

tctaagtagt tgattcatag tgactgcata tgttgtgttt tacagtatta tgtagtctgt 840

tttttatgca aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca 900

gcttttttat actaagttgg cattataaaa aagcattgct tatcaatttg ttgcaacgaa 960

caggtcacta tcagtcaaaa taaaatcatt atttgatttc aattttgtcc cactccctgc 1020

ctctgtcatc acgatactgt gatgccatgg tgtccgactt atgcccgaga agatgttgag 1080

caaacttatc gcttatctgc ttctcataga gtcttgcaga caaactgcgc aactcgtgaa 1140

aggtaggcgg atccccttcg aaggaaagac ctgatgcttt tcgtgcgcgc ataaaatacc 1200

ttgatactgt gccggatgaa agcggttcgc gacgagtaga tgcaattatg gtttctccgc 1260

caagaatctc tttgcattta tcaagtgttt ccttcattga tattccgaga gcatcaatat 1320

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Claims (9)

1. A DNA cloning method, comprising the steps of:

(1) Providing a double-stranded linearized vector, a double-stranded target gene fragment and a pair of primers, wherein the primers comprise an upstream primer and a downstream primer, two ends of the upstream primer are respectively provided with a pairing region which is complementary to the linearized vector and one end of the target gene fragment in the 3 'to 5' direction, and two ends of the downstream primer are respectively provided with a pairing region which is complementary to the linearized vector and the other end of the target gene fragment in the 3 'to 5' direction;

(2) Under the action of exonuclease, base groups at the 5 'ends of two ends of a double chain of the linearized vector and the target gene fragment are sequentially cut off, a single-stranded region at the 3' end is exposed for complementary pairing with a primer, and the single-stranded region is extended under the action of DNA polymerase after complementary pairing, so that the two ends of the linearized vector and the target gene fragment are provided with pairing regions;

(3) And (3) under the action of exonuclease, sequentially cutting bases at the 5 'ends of the double-stranded vector and the target gene fragment with the pairing regions at the two ends obtained in the step (2), exposing the single-stranded region at the 3' end for complementary pairing between the linearization vector with the pairing region and the target gene fragment, extending under the action of DNA polymerase after complementary pairing, and finally connecting into circular DNA under the action of ligase to obtain a cloning product of cloning the target gene fragment into the linearization vector.

2. The DNA cloning method of claim 1, wherein the steps (2) and (3) adopt a one-pot reaction, and the double-stranded linearized vector, the double-stranded target gene fragment and a pair of primers in the step (1), exonuclease, DNA polymerase, ligase and dNTPs are added into the reaction system at the same time.

3. The DNA cloning method of claim 2, wherein the exonuclease is T7 exonuclease; the DNA polymerase is at least one of Taq DNA polymerase, pfu DNA polymerase or KOD DNA polymerase; the ligase is Taq DNA ligase.

4. The method for cloning DNA according to claim 3, wherein the reaction system is that when the steps (2) and (3) are carried out by a one-pot reaction method:

the deficit was made up with ddH 2O.

5. The DNA cloning method of claim 3, wherein the reaction temperature of the steps (2) and (3) is 45 ℃ and the reaction time is 30min when the one-pot reaction is adopted.

6. The method for cloning DNA according to claim 1, wherein the double-stranded linearized vector obtained in step (1) is obtained by restriction endonuclease cleavage of a circular DNA vector.

7. The method for cloning a DNA according to claim 1, wherein the double-stranded target gene fragment in the step (1) is obtained by restriction enzyme digestion of a circular DNA on which the target gene is cloned.

8. The DNA cloning method of claim 1, wherein the length of the sequence of the pairing region between the two ends of the upstream primer and the downstream primer in step (1) and the linearized vector and the target gene fragment is 18bp to 30bp.

9. The DNA cloning method of claim 1, wherein the recombinant Escherichia coli containing the correct cloning product is obtained by screening after transforming competent Escherichia coli cells with the cloning product of step (3).

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