CN110863004A - Yeast two-hybrid vector, construction method and application thereof in protein interaction - Google Patents
Yeast two-hybrid vector, construction method and application thereof in protein interaction Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a yeast two-hybrid vector, a construction method and application thereof in protein interaction. The yeast two-hybrid vector comprises: pNC-GADT7 vector and pNC-GBKT7 vector; the pNC-GADT7 vector inserts a Nimble Cloning frame into the pGADT7 vector at the Cloning site; the pNC-GBKT7 vector was inserted into a Nimble Cloning frame at the Cloning site of the pGBKT7 vector. The yeast two-hybrid vector of Nimble Cloning is used to facilitate the Cloning of target genes into the vector. Has the advantages of simple operation, high cloning efficiency, standardized cloning and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a yeast two-hybrid vector, a construction method and application thereof in protein interaction.
Background
The interaction between protein and protein constitutes one main component of cell biochemical reaction network, and the protein-protein interaction network and transcription regulation network have important significance in regulating cell and its signal.
The yeast two-hybrid system is an important method currently widely used in protein interactomics research. The principle is that after the target protein and the bait protein are specifically combined, the bait protein is combined with a promoter of the reporter gene to start the expression of the reporter gene in the yeast cell, if an expression product of the reporter gene is detected, the interaction between the target protein and the reporter gene is indicated, otherwise, the interaction between the target protein and the bait protein is absent. The technology can be used for researching the interaction between proteins on a large scale after being subjected to micro-quantification and array. In actual work, a single hybridization system, a triple hybridization system, a reverse hybridization system, and the like have been developed as needed. The yeast two-hybrid vector used at present is mainly based on two cloning technologies of enzyme digestion-connection and Gateway, and has the problems of complicated operation or expensive reagent and the like.
Therefore, the yeast two-hybrid vector which is simple to operate, high in cloning efficiency and capable of being cloned in a standardized manner has important practical significance.
Disclosure of Invention
In view of the above, the present invention provides a yeast two-hybrid vector, a construction method and an application thereof in protein interaction. The invention constructs a set of yeast two-hybrid vectors based on Nimble Cloning. By using the set of vectors, the target gene can be cloned to the vector by using a NimbleCloning cloning technology, and then the target gene is used for protein interaction research.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a yeast two-hybrid vector comprising: pNC-GADT7 vector and pNC-GBKT7 vector;
the pNC-GADT7 vector inserts a Nimble Cloning frame into the pGADT7 vector at the Cloning site;
the pNC-GBKT7 vector is inserted into a Nimble Cloning frame at the Cloning site of a pGBKT7 vector;
the Nimble Cloning frame is formed by sequentially connecting a first linker sequence shown as SEQ ID No.1, a SfiI enzyme cutting site, a ccdB gene, the SfiI enzyme cutting site and a second linker sequence shown as SEQ ID No. 2.
On the basis of the research, the invention also provides a construction method of the yeast two-hybrid vector, which comprises the following steps:
step 1: respectively amplifying sequences at Cloning sites of yeast two-hybrid vectors pGADT7 and pGBKT7 to obtain a Nimble Cloning frame AD-NC inserted into pGADT7 and a Nimble Cloning frame BD-NC inserted into pGBKT 7;
step 2: taking pGADT7, carrying out NdeI and XhoI double enzyme digestion, mixing the recovered linearized vector pGADT7 with AD-NC, transforming the competence of escherichia coli DB3.1 by a reaction product, selecting a single clone, carrying out PCR (polymerase chain reaction) and sequencing identification, and obtaining a yeast double-hybrid vector pNC-GADT7 containing an NC cloning frame;
and step 3: taking pGBKT7, carrying out NdeI and PstI double enzyme digestion, mixing the recovered linearized vector pGBKT7 with BD-NC, transforming the competence of escherichia coli DB3.1 by a reaction product, selecting a single clone, carrying out PCR (polymerase chain reaction) and sequencing identification, and obtaining a yeast double-hybrid vector pNC-GBKT7 containing an NC cloning frame.
In some embodiments of the present invention, the primer set amplified in step 1 to obtain the NimbleCloning cloning frame AD-NC inserted into pGADT7 includes a forward primer NC-ADF shown in SEQ ID No.3 and a reverse primer NC-ADR shown in SEQ ID No. 4;
the primer set amplified in step 1 to obtain the Nimble Cloning frame BD-NC inserted into pGBKT7 comprises a forward primer NC-BDF shown as SEQ ID No.5 and a reverse primer NC-BDR shown as SEQ ID No. 6.
In some embodiments of the invention, the conditions for the amplification in step 1 are: 2 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 60 seconds; final extension at 72 ℃ for 5 min.
The invention also provides the application of the yeast two-hybrid vector or the yeast two-hybrid vector obtained by the construction method in protein interaction research.
On the basis of the research, the invention provides an identification method of different protein interaction, the yeast two-hybrid vector or the yeast two-hybrid vector obtained by the construction method is used as a cloning vector, and the steps comprise:
and 3, transforming the yeast competent cells and culturing by using a selective medium, wherein if the transformed strains can grow, the interaction between the different proteins is shown, otherwise, the interaction between the different proteins cannot be realized.
In some embodiments of the present invention, the identification method is provided wherein the reaction in step 2 is a reaction at 50 ℃ for 1 hour;
the Nimble Mix contains SfiI restriction endonuclease, T5 exonuclease and Phusion DNA polymerase;
the competent cell in the step 2 is an Escherichia coli DH5 α competent cell;
the competent cells in step 3 are yeast AH109 competent cells.
In some embodiments of the invention, the genes include murine p53 gene, SV40large T-antigen gene and Human lamin C gene;
the primer group for amplifying the murine p53 gene comprises a forward primer BD-53F shown as SEQ ID No.7 and a reverse primer BD-53R shown as SEQ ID No. 8;
the primer group for amplifying the Human lamin C gene comprises a forward primer BD-LamF shown as SEQ ID No.9 and a reverse primer BD-LamR shown as SEQ ID No. 10;
the primer group for amplifying the SV40large T-antigen gene comprises a forward primer AD-TF shown as SEQ ID No.11 and a reverse primer AD-TR shown as SEQ ID No. 12.
In some embodiments of the invention, the reaction conditions for the amplification in step 1 are: 2 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 90 seconds; final extension at 72 ℃ for 5 min.
In some embodiments of the invention, the selection medium in step 3 is SD/-Leu/-Trp and/or SD/-Leu/-Trp/-His/-Ade.
Specifically, recovered murine p53 and Human lamin C are respectively mixed with pNC-GBKT7, SV40large T-antigen is mixed with pNC-GADT7, Nimble Mix (Hainan Yitian biological Co., Ltd.) is added, reaction is carried out at 50 ℃ for 1h, a reaction product is transformed into escherichia coli DH5 α competence, a single clone is selected for PCR and sequencing identification, and the obtained recombinant plasmids are respectively named as pNC-BD-53, pNC-BD-Lam and pNC-AD-T.
The plasmid combinations pNC-AD-T and pNC-BD-53, pNC-AD-T and pNC-BD-Lam, and pAD-T and pBD-53, pAD-T and pBD-Lam for comparison were used to transform yeast AH109 competent cells. The conversion steps are as follows: taking the competent cells melted on 100 mul of ice, sequentially adding 0.5-1 mul of precooled target plasmid, 10 mul of salmon sperm DNA and 500 mul of PEG/LiAc, sucking and beating for several times, uniformly mixing, and carrying out water bath at 30 ℃ for 30min (turning for 6-8 times and uniformly mixing when 15 min); placing the tube in 42 deg.C water bath for 15min (turning 6-8 times when 7.5 min); centrifuging at 5000rpm for 40s and discarding the supernatant, re-suspending ddH2O 400 μ l, and centrifuging for 30s and discarding the supernatant; 100 μ l of ddH2O 100 were resuspended, 50 μ l of each of SD/-Leu/-Trp-coated and SD/-Leu/-Trp/-His/-Ade-coated plates were plated and incubated at 29 ℃ for 48-96 h. The transformation results are shown in FIG. 5, 4 combinations of transformed strains were able to grow on the SD/-Leu/-Trp plates with no difference between the NimbleCloning-based pNC combination and the control combination; on the four-deficient SD/-Leu/-Trp/-His/-Ade plates, the transformed strains of pNC-AD-T and pNC-BD-53, and pAD-T and pBD-53 were able to grow, indicating that the target proteins SV40large T-antigen and murine p53 interact, whereas the transformed strains of pNC-AD-T and pNC-BD-Lam, and pAD-T and pBD-Lam were not able to grow, indicating that the target proteins SV40large T-antigen and Human lamin C are not able to interact.
The invention constructs a set of yeast two-hybrid vector based on Nimble Cloning, so as to facilitate the Cloning of target genes into the vector. Has the advantages of simple operation, high cloning efficiency, standardized cloning and the like.
The main difference between the Nimble Cloning and Gibson Cloning is that the Nimble Cloning uses the circular plasmid directly, whereas the Gibson Cloning requires the circular plasmid to be linearized and recovered before it can be used in the Cloning reaction. The Cloning efficiency of both Cloning methods is shown in FIG. 6, and among the clones of 3 target genes, the Cloning efficiency of Nimble Cloning is higher than that of Gibson clone. Therefore, the use of yeast two-hybrid vector based on Nimble Cloning not only simplifies the operation but also improves the Cloning efficiency.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the amplification of NC cloning boxes; wherein, M: DNA marker; 1: AD-NC; 2: BD-NC;
FIG. 2 shows a map of pNC-GADT 7; wherein, the NC Frame is an NC Cloning Frame suitable for Nimble Cloning;
FIG. 3 shows a map of pNC-GBKT 7; wherein, the NC Frame is an NC Cloning Frame suitable for Nimble Cloning;
FIG. 4 shows amplification of genes for yeast two-hybrid; wherein, 1: murine p 53; 2: human lamin C; 3: SV40large T-antigen; m: DNA marker;
FIG. 5 shows a yeast two-hybrid vector based on Nimble Cloning for protein interaction studies;
FIG. 6 shows a comparison of Cloning efficiency of two Cloning methods based on yeast two-hybrid vector of Nimble Cloning.
Detailed Description
The invention discloses a yeast two-hybrid vector, a construction method and application thereof in protein interaction, and a person skilled in the art can realize the method by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention constructs a set of yeast two-hybrid vectors based on Nimble Cloning. Using this set of vectors, the target gene can be cloned into the vector using the Nimble Cloning technique.
Specifically, the yeast two-hybrid vectors pNC-GADT7 and pNC-GBKT7 based on Nimble Cloning are obtained by using yeast two-hybrid vectors pGADT7 and pGBKT7 as a framework, designing two pairs of primers, respectively carrying out PCR amplification on NC Cloning frames, and respectively inserting the NC Cloning frames into multiple Cloning sites of pGADT7 and pGBKT 7.
By using the set of vectors, target genes can be cloned to the vectors by using a Nimble Cloning technology, and then the target genes are used for protein interaction research.
FIG. 5 shows the case of parallel use (comparison) of the vectors of the present patent application with the present vectors, and as a result, their performance in protein interaction studies is consistent, and the purpose is to demonstrate that the performance of the modified vectors of the present patent vectors has no effect on protein expression. The vectors of the present patent application are characterized in that they have a Cloning frame sequence of Nimble Cloning, and a target gene can be cloned into the vectors using Nimble Cloning. The vector of the patent application and the existing vector have the advantages of cloning methods, including simple operation, high cloning efficiency, standardized cloning and the like.
The yeast two-hybrid vector, the construction method and the raw materials and reagents used in the application of the yeast two-hybrid vector in protein interaction are all commercially available.
The invention is further illustrated by the following examples:
example 1 construction of yeast two-hybrid vector based on Nimble Cloning
The Nimble Cloning frame provided by the invention is formed by sequentially connecting a first connector sequence shown as SEQ ID No.1, a SfiI enzyme cutting site, a ccdB gene, the SfiI enzyme cutting site and a second connector sequence shown as SEQ ID No. 2.
1.1 amplification of the NC cloning cassette
Two pairs of primers (Table 1) were designed based on the sequences at the cloning sites of the yeast two-hybrid vectors pGADT7 and pGBKT7, wherein the primer pair NC-ADF and NC-ADR was used to amplify the NC cloning frame AD-NC inserted into pGADT7 and the primer pair NC-BDF and NC-BDR was used to amplify the NC cloning frame BD-NC inserted into pGBKT 7. PCR amplification was performed using plasmid pNC-UC containing the NC cloning cassette as a template and high fidelity enzyme PrimeSTAR (Dalibao organism) under the following reaction conditions: 2 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 60 seconds; final extension at 72 ℃ for 5 min. The PCR products were detected by agarose gel electrophoresis, and the results are shown in FIG. 1. Electrophoresis results show that amplification yielded 2 bands between 500bp and 750bp, consistent with the expected size. The amplified band was recovered and recovered by agarose gel recovery kit (Dalianbao biosome) according to the instructions.
TABLE 1 primer sequences
1.2 insertion of NC cloning cassette into vector cloning site
pGADT7 was double digested with NdeI and XhoI and pGBKT7 was double digested with NdeI and PstI at 37 ℃ for 1 h. And (4) carrying out agarose gel electrophoresis and recovery after enzyme digestion. The recovered linearized vector pGADT7 was mixed with AD-NC and pGBKT7 with BD-NC, and Gibson cloning reaction solution (NEB) was added thereto at 50 ℃ for 1 hour. And transforming the reaction product into escherichia coli DB3.1 competence, selecting a single clone, and performing PCR and sequencing identification to obtain yeast double hybrid vectors pNC-GADT7 and pNC-GBKT7 containing NC cloning frames. The maps of pNC-GADT7 and pNC-GBKT7 are shown in FIGS. 2 and 3, respectively.
Example 2 application of yeast two-hybrid vector based on Nimble Cloning in protein interaction research
The invention selects the commonly used contrast protein in a yeast two-hybrid system, namely murine p53 and SV40large T-antigen interaction as positive; human lamin C and SV40large T-antigen did not interact as negative.
2.1 amplification of the target Gene
3 pairs of primers (Table 1) were designed based on the nucleic acid sequences of 3 genes, murine p53, Human lamin C and SV40large T-antigen, for positive and negative control in yeast two-hybrid experiments, wherein BD-53F and BD-53R were used to amplify the murine p53 gene cloned to pNC-GBKT7, BD-LamF and BD-LamR were used to amplify the Human lamin C gene cloned to pNC-GBKT7, and AD-TF and AD-TR were used to amplify the SV40large T-antigen gene cloned to pNC-GADT 7. The 3 pairs of primers respectively take plasmids pGBKT7-53(pBD-53), pGBKT7-Lam (pBD-Lam) and pGADT7-T (pAD-T) containing target genes as templates, and use high fidelity enzyme PrimeSTAR (Dalibao organisms), and the reaction conditions are as follows: 2 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 90 seconds; final extension at 72 ℃ for 5 min. The PCR products were detected by agarose gel electrophoresis, and the results are shown in FIG. 4. Electrophoresis results show that the amplification obtained a target band that corresponded to the expected size. And recovering the amplified band for subsequent cloning reaction.
2.2 cloning of the target Gene into the vector
Since pNC-GADT7 and pNC-GBKT7 are vectors based on Nimble Cloning, the target gene can be cloned to the vector by adopting a simple and efficient Nimble Cloning technology, the recovered murine p53 and Humanlamin C are respectively mixed with pNC-GBKT7, SV40large T-antigen is mixed with pNC-GADT7, Nimble Mix (Hainan Yida biology, Ltd.) is added, reaction is carried out at 50 ℃ for 1h, reaction products are transformed into escherichia coli DH5 α competence, single clones are selected for PCR and sequencing identification, and the obtained recombinant plasmids are respectively named as pNC-BD-53, pNC-BD-Lam and pNC-AD-T.
2.3 transformation of Yeast
The plasmid combinations pNC-AD-T and pNC-BD-53, pNC-AD-T and pNC-BD-Lam, and pAD-T and pBD-53, pAD-T and pBD-Lam for comparison were used to transform yeast AH109 competent cells. The conversion steps are as follows: taking the competent cells melted on 100 mul of ice, sequentially adding 0.5-1 mul of precooled target plasmid, 10 mul of salmon sperm DNA and 500 mul of PEG/LiAc, sucking and beating for several times, uniformly mixing, and carrying out water bath at 30 ℃ for 30min (turning for 6-8 times and uniformly mixing when 15 min); placing the tube in 42 deg.C water bath for 15min (turning 6-8 times when 7.5 min); centrifuging at 5000rpm for 40s, discarding the supernatant, ddH2O400 mul heavy suspension, centrifugation for 30s and abandoning the supernatant; ddH2Mu.l of the suspension was resuspended, 50. mu.l of each of the SD/-Leu/-Trp-coated and SD/-Leu/-Trp/-His/-Ade-coated plates were incubated at 29 ℃ for 48-96 h. The transformation results are shown in FIG. 5, and 4 combinations of transformed strains were able to grow on the SD/-Leu/-Trp plates with no difference between the Nimble Cloning based pNC combination and the control combination; on four-deficient SD/-Leu/-Trp/-His/-Ade plates, pNC-AD-T and pNC-BD-53, and pAD-T and pBD-53, showed growth of the transformed strains, indicating interaction of the target proteins SV40large T-antigen and murine p53, while pNC-AD-T and pNC-BD-Lam, and pAD-T and pBD-Lam, showed growth of the transformed strains, indicating interaction of the target proteins SV40large T-antigen and Human lamin C.
Example 3 comparison of Cloning efficiency of two Cloning methods based on yeast two-hybrid vector of Nimble Cloning
The method comprises the steps of utilizing yeast two-hybrid vectors pNC-GADT7 and pNC-GBKT7 based on Nimble Cloning, selecting Murine p53, Human lamin C and SV40large T-antigen as target genes, Cloning the target genes into the vectors by respectively adopting Nimble Cloning and currently widely used Gibson Cloning, comparing the Cloning efficiency of the target genes, wherein the design of primers of the two Cloning methods is the same and is the sequence listed in Table 1. the operation of the Nimble Cloning is the same as that of 2.2. the operation of the Gibson Cloning is that firstly, the vectors pNC-GADT7 and pNC-GBKT7 are cut by SfiI under the condition of 50 ℃ for 1h, and then, agarose gel electrophoresis is carried out for recovery, the recovered linearized vectors are respectively mixed with target gene fragments, added with Gibson (NEB) for 50 ℃ reaction for 1h, the reaction products are transformed into Escherichia coli 5 α, the number of the strains is counted after culture, the calculation of the Cloning efficiency of the Cloning and the Cloning efficiency of the Gibson the yeast two Cloning methods are respectively used for Cloning, and the Cloning of the Gibson the yeast two Cloning is carried out by the Cloning methods, so that the Cloning of the Cloning is carried out by using the yeast two Cloning methods, the Cloning of the Cloning method, and the Cloning of the Nimble Cloning is.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (10)
1. A yeast two-hybrid vector comprising: pNC-GADT7 vector and pNC-GBKT7 vector;
the pNC-GADT7 vector inserts a Nimble Cloning frame into the pGADT7 vector at the Cloning site;
the pNC-GBKT7 vector is inserted into a Nimble Cloning frame at the Cloning site of a pGBKT7 vector;
the Nimble Cloning frame is formed by sequentially connecting a first linker sequence shown as SEQ ID No.1, a SfiI enzyme cutting site, a ccdB gene, the SfiI enzyme cutting site and a second linker sequence shown as SEQ ID No. 2.
2. The method for constructing a yeast two-hybrid vector according to claim 1, comprising the steps of:
step 1: respectively amplifying sequences at Cloning sites of yeast two-hybrid vectors pGADT7 and pGBKT7 to obtain a Nimble Cloning frame AD-NC inserted into pGADT7 and a Nimble Cloning frame BD-NC inserted into pGBKT 7;
step 2: taking pGADT7, carrying out NdeI and XhoI double enzyme digestion, mixing the recovered linearized vector pGADT7 with AD-NC, transforming the competence of escherichia coli DB3.1 by a reaction product, selecting a single clone, carrying out PCR (polymerase chain reaction) and sequencing identification, and obtaining a yeast double-hybrid vector pNC-GADT7 containing an NC cloning frame;
and step 3: taking pGBKT7, carrying out NdeI and PstI double enzyme digestion, mixing the recovered linearized vector pGBKT7 with BD-NC, transforming the competence of escherichia coli DB3.1 by a reaction product, selecting a single clone, carrying out PCR (polymerase chain reaction) and sequencing identification, and obtaining a yeast double-hybrid vector pNC-GBKT7 containing an NC cloning frame.
3. The method of claim 2, wherein the primer set amplified in step 1 to obtain the Nimble Cloning frame AD-NC inserted into pGADT7 includes a forward primer NC-ADF shown in SEQ ID No.3 and a reverse primer NC-ADR shown in SEQ ID No. 4;
the primer set amplified in step 1 to obtain the Nimble Cloning frame BD-NC inserted into pGBKT7 comprises a forward primer NC-BDF shown as SEQ ID No.5 and a reverse primer NC-BDR shown as SEQ ID No. 6.
4. The method of claim 2, wherein the amplification conditions in step 1 are: 2 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 60 seconds; final extension at 72 ℃ for 5 min.
5. Use of the yeast two-hybrid vector according to claim 1 or the yeast two-hybrid vector obtained by the construction method according to any one of claims 2 to 4 in protein interaction research.
6. The method for identifying the interaction between different proteins, comprising the steps of using the yeast two-hybrid vector according to claim 1 or the yeast two-hybrid vector obtained by the construction method according to any one of claims 2 to 4 as a cloning vector:
step 1, amplifying genes of different proteins respectively to obtain the genes of different proteins;
step 2, mixing the genes of different proteins, the cloning vector and the Nimble Mix respectively, reacting, converting reaction products into competent cells, selecting monoclone, and performing PCR and sequencing identification to obtain recombinant plasmids containing different protein genes respectively;
and 3, transforming the yeast competent cells and culturing by using a selective medium, wherein if the transformed strains can grow, the interaction between the different proteins is shown, otherwise, the interaction between the different proteins cannot be realized.
7. The method of claim 6, wherein the reaction in step 2 is a reaction at 50 ℃ for 1 hour;
the Nimble Mix contains SfiI restriction endonuclease, T5 exonuclease and Phusion DNA polymerase;
the competent cell in the step 2 is an Escherichia coli DH5 α competent cell;
the competent cells in step 3 are yeast AH109 competent cells.
8. The method of claim 6 or 7, wherein said genes comprise murine p53 gene, SV40large T-antigen gene and Human lamin C gene;
the primer group for amplifying the murine p53 gene comprises a forward primer BD-53F shown as SEQ ID No.7 and a reverse primer BD-53R shown as SEQ ID No. 8;
the primer group for amplifying the Human lamin C gene comprises a forward primer BD-LamF shown as SEQ ID No.9 and a reverse primer BD-LamR shown as SEQ ID No. 10;
the primer group for amplifying the SV40large T-antigen gene comprises a forward primer AD-TF shown as SEQ ID No.11 and a reverse primer AD-TR shown as SEQ ID No. 12.
9. The method of any one of claims 6 to 8, wherein the amplification in step 1 is performed under reaction conditions selected from the group consisting of: 2 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 90 seconds; final extension at 72 ℃ for 5 min.
10. The method of any one of claims 6 to 9, wherein the selection medium in step 3 is SD/-Leu/-Trp and/or SD/-Leu/-Trp/-His/-Ade.
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