KR20000022394A - Method for effecting site-directed mutagenesis - Google Patents

Abstract

PURPOSE: It is concerned that a method for performing site-directed mutagenesis used in genetic engineering techniques, more easily and efficiently, and a kit for the use in the method CONSTITUTION: A method for effecting site-directed mutagenesis characterized by involving the step of effecting PCR by using a double-stranded DNA vector having one or more amber codons carrying a DNA fragment inserted thereinto and serving as the target in the site-directed mutation and at least two types of selective primers; and a site-directed mutagenesis kit for effecting this method characterized by containing primers for the reversion of the amber codons. The method and the kit are useful in more conveniently and quickly effecting the site-directed mutagenesis in the fields of genetic engineering and protein engineering. By using the method and the kit, it is possible to obtain a gene wherein mutagenesis has been efficiently effected at the desired site merely by transforming a PCR product obtained by PCR into a host.

Description

Site specific mutation introduction method

In recent years, in the field of genetic engineering, it is difficult to obtain a large amount of gene products because it only depends on the technique of cloning the expressed gene, and few successful cases have been obtained. For this reason, in order to increase the expression level of cloned genes, a technique of matching the frame and a technique of changing the nucleotide sequence near the start codon without changing the amino acid sequence (silent mutation) is a minimum necessary technique. In addition, when the protein is an enzyme, in order to perform cloning of the gene and to produce a protein useful as an expressed protein, the specificity of the protein, for example, may be changed by changing or deleting amino acids by changing the base sequence of the codon. Techniques for changing the optimal pH, stability, substrate specificity, Km value, etc. are important and necessary.

As described above, a method of changing a specific nucleotide sequence in a cloned gene as desired, i.e., site-specific mutagenesis, is a method of analyzing the structure and function of various regulatory regions on a gene including RNA, and using a recombinant DNA technique. It is essential for the performance of engineering applications. In addition, in the field of protein engineering, site-directed mutagenesis methods are more important in terms of faster and more accurate studies by introducing and deleting mutations at the DNA level.

Typically, site specific mutagenesis methods include, for example, the following processes:

(1) First, a target DNA into which a mutation is to be introduced is inserted into a vector. Then, when using double-stranded plasmid DNA, the complementary strand of the target DNA is dissociated by heat denaturation, or single-stranded DNA is prepared using an M13 phage vector;

(2) An oligonucleotide (mutant introduction primer) designed to introduce a desired mutation is annealed with the single-stranded DNA. Thereafter, the DNA polymerase and the DNA ligase are reacted to synthesize complementary DNA in an in vitro system;

(3) E. coli (Escherichia coli) is transformed with the DNA obtained in the above (2), and the clones into which the mutation is introduced are selected.

However, when only the above steps are carried out, the ratio of the variant to the parental DNA (parent DNA) is extremely low, so it is necessary to effectively select clones generated by conjugation to the mutagenesis primer. Therefore, in the process of said (3), the selective removal system is used so that the clone which has a mother DNA may not grow.

Examples of such systems include methods using amber mutations (amber codons). Nucleic Acids Research, 12, No. 24, pp.9441-9456 (1984); Methods using restriction enzyme sites [Analytical Biochemistry, 200, pp. 81-88 (1992); Gene, 102, pp. 67-70 (1991); And methods using dut (dUTPase) mutations and ung (uracil DNA glycosylase) mutations [Proceedings of the National Academy of Sciences of the USA, 82, pp. 488-492 (1985)]. However, in the above-described mutagenesis method, these processes are complicated and time consuming. In addition, in the above method, the yield of the desired clone into which the mutation is introduced is low.

On the other hand, the site-specific mutation introduction method described in Japanese Patent Laid-Open No. 7-289262 is a method using an amber mutation using DNA polymerase and DNA ligase. Compared with the above method, even if the variants can be produced with high efficiency in the method described in Japanese Patent Laid-Open No. 7-289262, two-stage transformation into E. coli is required, and thus simple manipulation for practical purposes is required. Being disturbed

Recently, site-directed mutagenesis methods have been developed based on PCR technology that is widely used.

For example, using the three or more primers including the mutagenesis primer to synthesize a DNA chain to be introduced into the mutation; Then cutting the DNA chain expected to introduce the mutation with a restriction enzyme and ligation to another vector; And transforming host E. coli with the obtained vector. In addition, DNA polymers of PCR or Pyrococcus furiosus using two complementary mutagenesis oligonucleotides (mutagenic primers) that can hybridize with the double stranded DNA into which the mutations are to be introduced. Synthesis of the chain with Lager followed by cleavage of the resulting chain with the restriction enzyme DpnI, followed by transformation of the host E. coli with the product treated with DpnI, resulting in a DNA with the trade name Quick Change. (Quik Change ^™ ) site specific mutagenesis kit (manufactured by Stratagene) can be used. In addition, a class IIS restriction enzyme recognition site was added to the 5'-terminal side of each of the two mutagenesis oligonucleotides (mutation introduction primers), and the chain was synthesized by PCR. Methods are known in which DNA with introduced mutations can be obtained, including cutting the introduced DNA, ligation and transforming host E. coli (US Pat. No. 5,512,463).

As described above, conventional methods using DNA polymerase and DNA ligase require a number of transformation steps to immobilize the enzymatic reactions and mutation sites described above, which takes too much time, thereby improving efficiency. Difficult to promote In addition, methods using amber mutations or “dut” and “ung” mutations must isolate single stranded DNA. The method of removing restriction enzyme sites has the same problem as that includes restriction of available restriction enzymes.

In view of the above, a site-directed mutagenesis implementation method using a widely used PCR technique has recently been developed and practically applied. However, three or more primers are required, including a mutagenic primer and a primer including two or more mutagenic primers, a restriction enzyme reaction during the operation, and other operations are complicated. Do. Other various problems include extremely limited mutation efficiency due to incomplete restriction enzyme reactions.

Accordingly, it is an object of the present invention to provide a kit for carrying out a site-specific mutagenesis method and a site-specific mutagenesis method using a PCR method, which is simple and practical.

As a result of intensive research to develop an efficient and simple method for performing site-directed mutagenesis, the present inventors succeeded in obtaining a clone into which a target mutation was introduced by transforming Escherichia coli only once after PCR. The present invention has been completed based on this fact.

The present invention relates to a method for carrying out site-directed mutagenesis more easily and efficiently for use in genetic engineering techniques, and to a kit for use in the method.

1 is a schematic diagram showing the structure of plasmid pKF19k.

2 is a schematic diagram showing the structure of plasmid pKF19kM.

Accordingly, the gist of the present invention is as follows:

[1] a method comprising a double-stranded DNA vector having one or more amber codons, a vector obtained by introducing a target DNA fragment for site-directed mutagenesis, and PCR using two or more selection primers Site-specific mutagenesis implementation method characterized by;

[2] a method of performing site-specific mutagenesis according to the above [1], wherein the selection primer is a mutagenesis primer and an amber codon reversion primer;

[3] a method of performing site-directed mutagenesis according to the above [1] or [2], wherein the vector has a drug resistance gene containing one or more amber codons;

[4] further comprising the step of transforming a suppressor-free (Sup ^o ) host with a PCR product using the site-directed mutagenesis method according to any one of [1] to [3]. A method comprising;

[5] a method for performing site-specific mutagenesis according to the above [4], wherein the suppressor-free (Sup ^o ) host is E. coli;

[6] A kit for site-directed mutagenesis used in the method for performing site-directed mutagenesis according to any one of [1] to [5], characterized by comprising an amber codon reversion primer. A kit;

[7] A kit for site-directed mutagenesis according to the above [6], wherein the kit comprises a vector having one or more amber codons;

[8] A kit for site-directed mutagenesis according to [6] or [7], wherein the kit comprises a suppresso-free (Sup ^o ) host; And

[9] A kit for site-directed mutagenesis according to the above [8], wherein the suppressor-free (Sup ^o ) host is E. coli.

The present invention is described in detail below.

According to the present invention, PCR is performed using a double-stranded DNA vector having one or more amber codons and a vector obtained by inserting a target DNA fragment for site-specific mutagenesis, and two or more selection primers. A method comprising the steps of performing. As the selection primer used in the present invention, two or more primers composed of a mutagenesis primer for inducing a mutation at a desired position on a gene to be mutated may be used; As the amber codon reversion primer for amber codon reversion, an amber codon reversion primer arranged on the opposite chain of the mutagenic primer can be used. The term "two or more primers" as used herein refers to two or more selections described herein, including any set of selection primers as long as they include mutagenic primers, amber codon reversion primers, and two selection primers. Refers to the primer.

The double-stranded DNA vector having one or more amber codons used in the present invention may be any vector as long as there is no limitation to one or more amber codons and the site-specific mutagenesis method of the present invention. In such cases, it is preferable to use vectors with amber codon (s) on the drug resistance gene. The drug resistance gene is not particularly limited, but for example, kanamycin resistance gene, chloramphenicol resistance gene and the like are preferable. In other words, a vector having one or more amber codons on a gene of resistance, such as, for example, kanamycin or chloramphenicol gene, is preferably used. As an example, pKF19k (manufactured by Shoe Industries) can be used. Cassettes containing amber codons can be prepared to introduce the cassettes into the vector. By using such a vector, a PCR product is simply introduced into a suppressor-free host such as Escherichia coli, and the obtained host is cultured in agar medium containing a drug such as kanamycin, chloramphenicol, and the like. It is easy to see the return of the amber codon on the bed.

The kind of mutation which can be introduced by the site specific mutagenesis implementation method of this invention is not specifically limited. Examples thereof include base substitution, deletion and insertion. Although the variation size is not limited, it is preferable to change the length of the mutagenic primer according to the variation size. For example, when the mutation size is about 1 to 3 bases, the length of the mutagenic primer is preferably 20 bases. When the mutation size is 4 bases or more, it is preferable to use mutagenic primers up to about 30 bases, including about 15 base pairs on each of the 5'-side and the 3'-side with respect to the target mutation site. . It is also preferred that the 3'-terminus for mutagenesis is G or C, since the 3'-terminus serves as a starting point for the polymerase reaction. Considering these conditions, a mutagenic primer is prepared. In addition, the design and purity of the primer can be evaluated by confirming whether the target fragment is amplified by PCR using the primer by electrophoresis or the like. In addition, in relation to the number of the mutagenesis primers in the present invention, the target mutations can be introduced as one kind of mutagenesis primers. However, when introducing other mutations at one site, a plurality of mutagenesis primers can be used in the mixture to obtain a gene for introducing each desired mutation by one operation, depending on the purpose. For example, a base-substituted site-specific mutagenesis of nucleotide conversion from G to A, T, or C at one position results in the introduction of a mutation from G to A, T, or C, respectively. Three kinds of mutagenic primers may be prepared and then obtained by one-time operation using the mutagenic primers in a mixture.

In addition, the amber codon reversion primer is a primer which can return the amber codon part to a wild type etc. without being specifically limited. For example, if the amber codon is TAG, the amber codon can be returned using a primer prepared to convert the TAG to TCG or CTG. The length of the amber codon reversion primer is preferably changed according to the number of amber codons, PCR efficiency and the like. The length of the amber codon reversion primer is not particularly limited. Preferably from about 20 to 30 bases in length. It is also preferred that the 3'-terminus of the amber codon is G or C, as is the variant primer, because the 3'-terminus acts as a starting point for the polymerase reaction. In addition, the design and purity of the primer can be evaluated by confirming whether the target fragment is amplified by PCR using the primer by electrophoresis or the like. It is also preferable to use one kind of amber codon reversion primer. When two or more amber codons are present, it is preferable to prepare a amber codon reversion primer so that the amber codon can be returned by one kind of primer.

The concentration of each mutagenic primer and amber codon reversion primer in PCR is not particularly limited. It is necessary to study each optimal concentration in the reaction, which reduces the amount of amplification when the concentration of each primer is very low, and promotes non-specific reactions when the concentration is very high, resulting in some cases starting the specific amplification reaction. Because it can interfere. In general, PCR is preferably performed with the final concentration range of each primer being 0.1 to 1.0 μM.

The suppressor-free host referred to herein includes, but is not limited to, E. coli, for example E. coli MV1184 (manufactured by Takara Shuzo). Any host can be used so long as it does not have a suppression ability, and all such hosts are included within the scope referred to herein as suppressor-free hosts.

Site specific mutagenesis by the method of the invention can be carried out, for example, by the following steps:

(1) The target DNA is inserted into a double stranded DNA vector having one or more amber codons.

(2) the DNA-insertion vector prepared in (1) above, a mutagenic primer to introduce a mutation at a target site of the gene to be mutagenic, and an amber codon for the return of the mutant codon arranged in the opposite chain of the mutagenic primer PCR is performed by mixing the amber codon return primers.

3 is a PCR product suppressor-free (Sup ^o) is introduced into a host and screened for clones having the desired mutation.

By performing the above steps, a gene in which mutations are introduced at a target site can be obtained efficiently.

In the present invention, since the PCR product by PCR amplification acts as a long-chain primer, the PCR product promotes full-length DNA synthesis during the PCR process. In addition, when introduced directly into E. coli as a host, the PCR product cyclizes. In addition, when a clone including a gene having a mutation introduced at a target site is selected, the selection of the suppression system method simplifies the selection. Expressed correctly, amber codon reversion by introducing a PCR product into a host, e.g., Suppressor-free E. coli, culturing the host in a medium containing the drug for one of the drug resistance genes and confirming its growth. You can simply check In other words, because only amber codon-returned clones are selected, the grown cells are clones with genes that have had mutations introduced at the clone target site.

According to the method of the invention, the mutation can be introduced at any site on the gene to be mutated.

Conventional methods can be used to perform PCR by the method of the present invention, but the following cautions are necessary to avoid the introduction of erroneous bases by PCR, such as the addition of adenine (A) at the 3'-end. For sake.

(I) The gene to be mutated and inserted into the vector is preferably 2 kbp or less.

(II) The number of PCR cycles is preferably 20 to 30 cycles.

(III) The thermostable DNA polymerase used for PCR preferably has high amplification efficiency and low error degree. For example, a TaKaRa LA PCR kit (manufactured by Takara Shu Survey) can be used as a PCR kit. In addition, the thermally stable DNA polymerase can achieve high amplification efficiency and low error rate by performing PCR using TaKaRa Ex Taq (manufactured by Takara Shu investigation).

As a method for transforming a host such as Escherichia coli into a PCR product and another host, the calcium chloride method [Journal of Molecular Biology, 166, pp. 557-580 (1983)] and the electroporation method [Nucleic Acids Research, 16, pp. 6127-6145 (1988). With respect to the screen of the obtained transformant, a clone containing a gene having a high probability amber codon return, ie, a gene having a high mutation probability at the target site, in a medium containing a drug against one of the drug resistance genes Transformants can be selected by culturing and confirming their growth.

Since the present invention provides a simple site-specific mutagenesis implementation method using an amber codon, one of ordinary skill in the art can apply a stop codon other than an amber codon, for example, an oak codon or an opal codon, to the amber codon position of the present invention. Self-explanatory Therefore, the use of the ocher codon or the opal codon at the position of the amber codon in the site-specific mutagenesis implementation method of the present invention is actually included within the scope of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1

Single base-substituted site specific mutations

1. Preparation of pKF19kM

pKF19kM (FIG. 2) was prepared with the plasmid to be used in the examples below. This was obtained by converting G at position 30 to A downstream from the multicloning position in the lacZ 'gene of pKF19k (manufactured by Takara Shuzo, FIG. 1), and a kanamycin resistance gene comprising two amber codons. To have.

The lacZ 'gene has β-galactosidase activity when the lacZ' gene is introduced into a host cell with the lacZΔM15 genotype. Therefore, in the case of the lacZ 'gene without base substitution, the obtained growth colony is 5-bromo-4- as a substrate in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG, manufactured by Takara Shuzo). It reacts with chloro-3-indolyl-β-D-galactoside (X-Gal, manufactured by Takara Shuzo) to give a blue color. On the other hand, the variant lacZ 'gene with base substitution cannot exhibit inherent activity, so the growth colony obtained is white.

Using this phenomenon, the return efficiency of base substitution in the pKF19kM variant lacZ 'gene by site-specific mutagenesis to the active gene is calculated from the number of blue and white colonies.

2. Synthesis of oligonucleotides for mutagenicity (mutagenic primers) and oligonucleotides for double stranded amber codon reversion in kanamycin resistance gene

The 5'-terminal phosphorylated oligonucleotide represented by SEQ ID NO: 1 in the sequence listing (mut1) is synthesized as a transgenic oligonucleotide used for the reversion of the base substitution position in the variant lacZ 'gene (return from A to G). In other words, mut1 is designed to revert base substitution of the tenth C to obtain the active lacZ 'gene.

In addition, the 5'-terminal phosphorylated oligonucleotide represented by SEQ ID NO: 2 in the Sequence Listing is prepared as a primer (KQ2) for the reversion of double amber codons (two amber codons) in the kanamycin resistance gene. Precisely, within pKF19kM, the primers are designed to return amber codons (TAG) to the TTG at the 3rd to 5th positions of KQ2 and the CTG at the 9th to 11th positions.

3. PCR

The composition of the reaction mixture is shown in Table 1. PCR is performed using a thermal cycler. The reaction mixture is desalted by ethanol precipitation and concentrated. The resulting mixture is then suspended in 5 μl of sterile water.

pKF19kM 5 ng Primers (mut1 and KQ2) 5 pmol each final concentration TaKaRa Ex Taq (Takara Shu investigation manufacture) 5 U dNTP mixture 200 μM for each final concentration TAPS buffer (pH 9.3, 25 ° C) 25 mM KCl 50 mM MgCl ₂ 2 mM 2-mercaptoethanol 1 mM 50 μl total volume (with mineral oil) Caution: TAPS = N-tris (hydroxymethyl) methyl-3-aminopropanesulfonate

The cycle is run under conditions of 30 seconds at 94 ° C, 2 minutes at 55 ° C, and 2 minutes at 72 ° C. The conditions are repeated 20 cycles for Cycle I and 24 cycles for Cycle II. Additionally, after termination of PCR, each product of Cycles I and II was subjected to electrophoresis to confirm that the DNA fragments of the desired size were amplified.

4. Transformation

Suppressor-free E. coli MV1184 (manufactured by Takara Shuzo Co., Ltd.) is transformed by electroporation using 2 µl of each PCR product after ethanol precipitation. Subsequently, the resulting transformed cells were cultured in LB agar culture [bactotryptone (10 g), bactoist extract containing kanamycin (50 μg / ml), X-Gal (40 μg / ml) and IPTG (0.2 mM). (5 g), NaCl (5 g), distilled water (1 liter), pH (7.0)] and calculate the efficiency of site-specific mutagenesis in the resulting clones. Incidentally, it should be noted that when the variant portion in the variant lacZ 'gene is returned to the active lacZ' gene, the resulting colony will appear blue and white if the variant portion has not returned. The results are shown in Table 2.

Blue White Total (Blue / Total) X100 [%] Cycle I 586 314 900 65 Cycle II 2655 891 3546 75

In other words, clones returned to the active gene by site specific mutagenesis were obtained at 75% efficiency by Cycle II.

5. Analysis of site-specific mutation sites

Plasmid DNA was prepared by alkali lysis on each of the four blue colonies obtained in Example 4 Clause 4. The base sequence of each plasmid DNA is determined by dideoxy method, and then the mutation site is analyzed. As a result, G returned from A was observed at the 30th position downstream of the multicloning site in the lacZ 'gene, clearly indicating that site-specific mutations are induced.

Example 2

3 base- or 6 base-delete site specific mutagenesis

To delete 3 or 6 bases, kanamycin resistance gene with two amber codons and pKF19k with wild type lacZ 'gene were used.

1. Synthesis of Mutagenic Oligonucleotides

5'-terminal phosphorylated oligonucleotides are synthesized to lack the BamHI site and the BamHI-EcoRI site by 3-base or 6-base deletions, respectively, at the multicloning site of pKF19k. Oligonucleotides used for 3-base deletion (del1) are shown in SEQ ID NO: 3 in Sequence Listing. Oligonucleotides used for 6-base deletion (del2) are shown as SEQ ID NO: 4 in Sequence Listing. To be precise, del1 is designed to delete the base at position 12 between G and 13th C of SEQ ID NO: 3 in the sequence listing, and del2 is between 12th G and 13th T of SEQ ID NO: 4 in the sequence listing It is designed to delete the base of the position.

2. PCR and transformation

Depending on the composition of Table 1 (in the table, pKF19k was used instead of pKF19kM), 55 seconds at 94 ° C. for 30 seconds using the KQ2 primer used in Example 1 and the primers del1 and del2 prepared in Example 1, respectively 30 cycles of PCR are carried out with each cycle composed of conditions of 2 minutes at < RTI ID = 0.0 > The resulting PCR product is ethanol precipitated in the same manner as in Example 1. The precipitated PCR product is then suspended in 5 μl of sterile water and E. coli MV1184 is transformed with 2 μl of the obtained suspension. The resulting transformants are then dispersed in LB agar medium containing 50 μl / ml kanamycin and each 10 cells are selected in growth colonies. Thereafter, plasmid DNA was prepared in the same manner as in Example 5, and 5. Identification of the mutation is carried out by digesting each of the plasmid DNAs with restriction enzyme BamHI. In other words, the plasmid into which the mutation is introduced is not digested. The results are shown in Table 3.

Non-digested clone count assay cell number variation efficiency (%) del1 9 10 90

Mutations introduced into each of the PCR products using primers del1 and del2 had a mutation efficiency of 90%, and as a result of sequencing, it was confirmed that 3 or 6 bases were deleted from each PCR product.

Example 3

60 base-deletion site-specific mutagenesis

1. Synthesis of Mutagenic Oligonucleotides

A 5'-terminal phosphorylated oligonucleotide was synthesized such that the deletion resulted in a lack of the entire multicloning site (60 bases) of pKF19k (manufactured by Takara Shuzo). Oligonucleotide (del3) is shown as SEQ ID NO: 5 in Sequence Listing. In other words, del3 is designed to delete the entire multicloning site (60 bases) between the 10th T and 11th G positions of SEQ ID NO: 5 in the sequence listing.

2. PCR and transformation

Depending on the composition of Table 1 (in the table, pKF19k was used in place of pKF19kM), the KQ2 primer used in Example 1 and the primer del3 prepared in Example 1 were used at 50 ° C. for 30 seconds at 94 ° C. 30 cycles of PCR are performed as each cycle composed of conditions for 2 minutes and 2 minutes at 72 ° C. The resulting PCR product is ethanol precipitated in the same manner as in Example 1. The precipitated PCR product is then suspended in 5 μl of sterile water and E. coli MV1184 is transformed with 2 μl of the obtained suspension. The resulting transformants are then dispersed in LB agar medium containing 50 μl / ml kanamycin and each 20 cells are selected in growth colonies. Thereafter, plasmid DNA was prepared in the same manner as in Example 5, and 5. Identification of the mutation is carried out by digesting each of the plasmid DNAs with restriction enzymes EcoRI and HindIII. In other words, the plasmid into which the mutation is introduced is not digested. The results are shown in Table 4.

Non-digested clone count assay cell number variation efficiency (%) del3 20 20 100

Mutations introduced by PCR with primer del3 had a mutation efficiency of 100%, and as a result of sequencing, the entire multicloning site (60 bases) was deleted.

This result shows that deletion of a relatively long region of base can be easily performed.

Example 4

Deletion and insertion site specific mutations

1. Synthesis of Mutagenic Oligonucleotides

The 5′-terminal phosphorylated oligonucleotide is synthesized such that an additional EcoRI site is introduced at the 100 th position downstream from the EcoRI site within the polycloning site of pKF19k. Oligonucleotide eco1 is shown as SEQ ID NO: 6 in Sequence Listing. In other words, eco1 deletes the AAT base of the original sequence corresponding to the base between the 9th G and 14th C and inserts the EcoRI site GAATTC.

2. PCR and transformation

Depending on the composition of Table 1 (in the table, pKF19k was used in place of pKF19M), using the KQ2 primer used in Example 1 and the primer eco1 prepared in paragraph 1 of Example 4, at 55 ° C. for 30 seconds at 94 ° C. 24 cycles of PCR are performed with each cycle configured for 2 minutes and at 72 ° C. for 2 minutes. The obtained PCR product is ethanol precipitated in the same manner as in Example 1. The precipitated PCR product is then suspended in 5 μl of sterile water and E. coli MV1184 is transformed with 2 μl of the obtained suspension. The resulting transformants are then dispersed in LB agar medium containing 50 μl / ml kanamycin and each 12 cells are selected in growth colonies. Thereafter, plasmid DNA was prepared in the same manner as in Example 5, and 5. Confirmation of the mutation was carried out by digesting each of the plasmid DNAs with the restriction enzyme EcoRI, followed by electrophoresis to identify 100 base DNA fragments. In other words, the plasmid into which the mutation is introduced has two EcoRI sites at 100 base intervals, which can be identified by digesting with the restriction enzyme EcoRI. The results are shown in Table 5.

Identified 100 Base Fragment Number Assay Cell Number Variation Efficiency (%) eco1 12 12 100

The mutation introduced using the primer eco1 confirmed that the target variant plasmid was obtained with a mutation efficiency of 100%. As a result of sequencing, it was confirmed that the EcoRI site was introduced into the target site.

Example 5

Introduction of mutations into DNA fragments ligated to pKF19k

1. Ligation of E. coli Plasmid Vectors pBR322 and pKF19k

A vector was prepared in which pKF19k was digested with the restriction enzyme BamHI, and the E. coli plasmid pBR322 having a length of 4361 bases was produced by Takahashi; Gene, 22, pp. 277-280 (1983)] is introduced into the BamHI site of pKF19k digested above. The obtained vector was named pKB101. Site specific mutagenesis for the insert fragments is made using the vector described above.

2. Synthesis of Mutagenic Oligonucleotides

The 5′-terminal phosphorylated oligonucleotide is synthesized so that an additional BamHI site is introduced at the 400 th position downstream from the BamHI site of pBR322. Oligonucleotide (bam1) is shown as SEQ ID NO: 7 in Sequence Listing. In other words, bam1 is designed so that the AGCGCT of the original sequence is replaced with BamHI site GAATTC at a position between 16th G and 21st C of SEQ ID NO: 7 in the sequence listing.

3. PCR and transformation

According to the composition of Table 1 (in the table, pKB101 was used in place of pKF19M), using the KQ2 primer used in Example 1 and the primer bam1 prepared in 2 of Example 5, at 55 ° C. for 30 seconds at 94 ° C. 25 cycles of PCR are performed, with each cycle consisting of conditions for 30 seconds and 6 minutes at 72 ° C. The obtained PCR product is ethanol precipitated in the same manner as in Example 1. The precipitated PCR product is then suspended in 5 μl of sterile water and E. coli MV1184 is transformed with 2 μl of the obtained suspension. Next, the resulting transformants are dispersed in LB agar medium containing 50 μl / ml kanamycin and each six cells are selected in growth colonies. Thereafter, plasmid DNA was prepared in the same manner as in Example 5, and 5. Confirmation of the mutation was carried out by digesting each of the plasmid DNAs with restriction enzyme BamHI, followed by electrophoresis to identify 400 base DNA fragments. In other words, the plasmid into which the mutation is introduced has two BamHI sites at 400 base intervals, and the 400 base fragments can be identified by digesting with the restriction enzyme BamHI. The results are shown in Table 6.

Identified 400 Base Fragment Number Assay Cell Number Variation Efficiency (%) bam1 5 6 83

It was confirmed that the target mutant plasmid using the DNA fragment ligated into pKF19k was obtained at 80% or more mutation efficiency. As a result of sequencing, it was confirmed that the BamHI site was introduced into the target site.

Example 6

Preparation of kits for site specific mutagenesis

Kits for site-directed mutagenesis runs (20 batches) were constructed as a set of host E. coli, vectors and oligonucleotides used as controls (Table 7).

Μl KQ2 primer (for return of amber codon; 5 pmol / μl) 20mut1 primer (mutant primer; 5 pmol / μl) 5pKF19kM (5 ng / μl) 5pKF18k and pKF19k (10 OD / ml) each E. coli MV1184 ( Stored in 10% glycerol solution) 100 weeks; pKF18k and pKF19k are the shoe irradiated products.

Additionally, the kits in Table 7 can be combined with the PCR kits on sale.

In addition, when constructing a kit for site specific mutagenesis runs (20 batches), the kit includes a set of mixtures, dNTP mixtures, thermostable DNA polymerase to perform PCR (Table 8).

Μl TaKaRa Ex Taq (5 U / μl; manufactured by Takara Shuzo) 2010X Ex Taq Buffer (manufactured by Takara Shozo) 200dNTP mixture (2.5 mM each) 240KQ2 Primer (for return of amber codon; 5 pmol / μl) 20mut1 Primers (priming for mutagenesis; 5 pmol / μl) 5pKF19kM (5 ng / μl) 5pKF18k and pKF19k (10 OD / ml) 100 Escherichia coli MV1184 (stored in 10% glycerol solution) for 100 weeks; pKF18k and pKF19k are the shoe irradiated products.

In accordance with the present invention, site-directed mutagenesis implementation methods and kits are provided, which give simpler and faster utility in genetic and protein engineering. By using the method and kit of the present invention, a PCR product obtained by PCR can be easily transformed into a host to efficiently obtain a gene in which mutations are introduced at a target site.

[Sequence list]

SEQ ID NO: 1

Sequence length: 20

Sequence type: nucleic acid

Number of chains: 1 chain

Topology: Straight

Molecular Type: Other Nucleic Acids (Synthetic DNA)

GGGTTTTCCC AGTCACGACG

SEQ ID NO: 2

Sequence length: 20

Sequence type: nucleic acid

Number of chains: 1 chain

Topology: Straight

Molecular Type: Other Nucleic Acids (Synthetic DNA)

GATTGCGCCT GAGCGAGACG

SEQ ID NO: 3

Sequence length: 23

Sequence type: nucleic acid

Number of chains: 1 chain

Topology: Straight

Molecular Type: Other Nucleic Acids (Synthetic DNA)

CGGTACCCGG GGCTCTAGAG TCG

SEQ ID NO: 4

Sequence length: 23

Sequence type: nucleic acid

Number of chains: 1 chain

Topology: Straight

Molecular Type: Other Nucleic Acids (Synthetic DNA)

CGGTACCCGG GGTAGAGTCG ACC

SEQ ID NO: 5

Sequence length: 20

Sequence type: nucleic acid

Number of chains: 1 chain

Topology: Straight

Molecular Type: Other Nucleic Acids (Synthetic DNA)

GACGGCCAGT GTAATCATGG

SEQ ID NO: 6

Sequence length: 24

Sequence type: nucleic acid

Number of chains: 1 chain

Topology: Straight

Molecular Type: Other Nucleic Acids (Synthetic DNA)

GCTGGCGTGA ATTCAGCGAA GAGG

SEQ ID NO: 7

Sequence length: 30

Sequence type: nucleic acid

Number of chains: 1 chain

Topology: Straight

Molecular Type: Other Nucleic Acids (Synthetic DNA)

CCGAAAATGA CCCAGGGATC CGCCGGCACC

Claims (9)

A vector obtained by inserting a double-stranded DNA vector having at least one amber codon and a target DNA fragment for site-specific mutagenesis, and performing a PCR using two or more selection primers Method of implementing specific mutagenesis. The method of claim 1, wherein the selection primer is a mutagenesis primer and an amber codon reversion primer. 3. The method of claim 1 or 2, wherein said vector has a drug resistance gene comprising one or more amber codons. The method of any one of claims 1 to 3, further comprising the step of transforming the suppressor-free (Sup ^o ) host with a PCR product. 5. The method according to claim 4, wherein said suppress ^o -free host is Escherichia coli. Site-specific mutagenesis kits used in the site-specific mutagenesis implementation method according to any one of claims 1 to 5, characterized in that the kit comprises a amber codon return primer. 7. The site specific mutagenesis kit of claim 6, wherein said kit comprises a vector having at least one amber codon. Claim 6 or claim 7, wherein the kit of the suppressor-free (Sup ^o) site-directed mutagenesis kit, characterized in that it comprises a host. The site specific mutagenesis kit according to claim 8, wherein the suppressor-free (Sup ^o ) host is Escherichia coli.