JP3526326B2 - Site-directed mutagenesis method - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to site-directed mutagenesis used in genetic engineering.
s) is further simplified, and a kit used for the method.

[0002]

2. Description of the Related Art In recent years, site-directed mutagenesis is the minimum required technique for clarifying the relationship between the structure and function of a cloned gene in the field of genetic engineering, and also in the field of protein engineering. It is an indispensable technique for modifying proteins and changing their properties. Conventionally, site-directed mutagenesis has been performed, for example, by the following procedure: After inserting a DNA into which a desired mutation is introduced into a vector, in the case of a double-stranded plasmid DNA, it is heat-denatured to dissociate a complementary strand. Or a M13-based phage vector is used to prepare single-stranded DNA. The oligonucleotide (mutation-introducing primer) chemically synthesized according to the desired mutation is used as the single-stranded DNA described above.
After annealing, the double-stranded DNA which is complementary except for the desired mutation is prepared in an in vitro system by polymerase reaction and ligase reaction. Escherichia coli is transformed with the above DNA, and a clone having a mutation introduced therein is selected. However, since the mutation is introduced into only one of the complementary strands by such a procedure as it is, after introduction into Escherichia coli, clones inevitably have no mutation introduced through replication. As a result, the rate of obtaining clones having the mutation introduced therein is low. Therefore, in order to increase this ratio,
In the process of (1), clones into which the DNA without mutation has been introduced have been devised so as to be selectively removed so as not to grow. For example, amber mutation, different restriction modification systems, use of dut (dUTPase) and ung (uracil-DNA glycosidase) mutations, etc. However, such a site-directed mutagenesis method is considerably complicated in procedure and considerably laborious.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a more convenient site-directed mutagenesis method and a kit for carrying out the method.

[0004]

SUMMARY OF THE INVENTION The present invention will be summarized. The first invention of the present invention relates to a site-directed mutagenesis method.
A method for site-directed mutagenesis, which comprises introducing a mutation into Escherichia coli in which an NA repair system is induced , comprising:
Step of: (1) UV irradiation, alkylating agent, or DNA replication inhibition
Treating the host E. coli by agent, (2) (1) site-specific mutations guide host E. coli cells prepared in
Vector for insertion of target DNA and for mutation introduction
A step of introducing DNA, a step of culturing the transformant prepared in (3) and (2),
It is characterized by including. Also relates the second invention site-directed mutagenesis kit of the present invention, parts of the first invention
A kit for use in a position-specific mutation introduction method, comprising:
By UV irradiation, alkylating agents, or DNA replication inhibitors
It is characterized in that it contains re-treated E. coli .

The present inventors have found that Escherichia coli inducing a DNA repair system preferentially replicates only the strand of the plasmid which undergoes homologous recombination, and efficiently and site-specifically in host cells. We have developed a method to introduce mutations. Furthermore,
The present invention has been completed by constructing a kit for conveniently performing site-specific mutation by the method.

The present invention will be described in detail below. The host cell used in the present invention may be a cell having a established host-vector system such as a bacterium such as Escherichia coli or Bacillus subtilis in which a DNA repair system is induced, yeast, plant cell, animal cell and the like. However, E. coli, which is easy to handle, is preferably used.

Hereinafter, an example in which Escherichia coli is used as a host cell will be mainly described. The site-specific mutagenesis by the method of the present invention using Escherichia coli as a host cell can be performed, for example, by the following steps. (1) The host Escherichia coli is treated to induce a DNA repair system (SOS repair). (2) The vector into which the DNA that is the target of site-specific mutagenesis is inserted and the mutagenesis DNA are introduced into the host Escherichia coli prepared in (1). (3) The transformant prepared in (2) is cultured to replicate the plasmid. At this time, only the strand in which a mutation has been introduced by homologous recombination preferentially replicates. (4) A plasmid is extracted to obtain a DNA having a mutation introduced therein.

As the host Escherichia coli used in (1), a strain in which SOS repair normally functions is used, and preferably a recA ⁺ strain in which the recA gene, which is a typical gene involved in SOS repair, functions normally. To be recA
⁺ Strains include BMH71-18mutS, C60
0, JM101, RR1 and the like.

The treatment for inducing the repair system of DNA is, for example, a treatment for damaging DNA, and examples thereof include UV irradiation and treatment with a drug. Examples of the drug include alkylating agents such as methyl methanesulfonate and ethyl methanesulfonate, and DNA replication inhibitors such as nalidixic acid.

The vector used in (2) may be any vector such as pUC system, pBR system, M1 often used in E. coli.
Three types of vectors can be used.

The mutation-introducing DNA is a natural or artificial DNA containing a desired mutation, and for example, a single-stranded synthetic oligonucleotide can be used. The length of the oligonucleotide is not particularly limited as long as it can hybridize stably with the target sequence, but it is generally 20 mer or more. Long-chain DNA such as PCR product as mutation-introducing DNA
By using, it is possible to carry out site-directed mutagenesis with higher efficiency. For example, PCR is carried out using an oligonucleotide containing the mutation of interest and an oligonucleotide complementary to another part of the vector as a primer pair, and the PCR product is used as a mutation-introducing DNA. This makes it possible to easily prepare long-chain DNA that is difficult to chemically synthesize. The longer the length of the long chain DNA, the more stable the efficiency can be obtained. In this method, if hybridization is unstable with an oligonucleotide, and the desired mutation cannot be introduced because there is a site with high complementarity with the oligonucleotide other than the desired mutation-introducing site, E. coli with low transformation efficiency is selected. It is particularly effective when used.

As a method for introducing DNA into the host Escherichia coli, electroporation is preferably used because it is a method which can obtain high transformation efficiency.

Next, the plasmid is replicated in (3).
Usually, in such a case, it is considered that a plasmid into which a mutation has been introduced and a plasmid into which a mutation has not been introduced coexist in the same bacterial cell. However, according to the experiments conducted by the present inventors, only the strand having the mutation introduced therein was preferentially replicated, and the plasmid containing no mutation was not mixed in the same bacterial cell. Therefore, according to this method, it is not necessary to select only the plasmid into which a mutation has been introduced using amber mutation or the like as in the conventional method. For this reason, there is a wide range of selection of host E. coli and vectors that can be used.

In the method of the present invention, since the double-stranded vector is used as it is, it is not necessary to prepare single-stranded DNA by phage infection or denaturation treatment as in the conventional case. Further, there is no need for an annealing step or a reaction step using an enzyme catalyst such as a polymerase reaction or a ligase reaction. Further, as described above, the step of selecting only the mutated plasmid is not required. Site-directed mutagenesis can also be introduced by in vitro homologous recombination with proteins involved in DNA repair. For example, in the presence of ATP, RecA protein, which is one of the typical proteins involved in DNA repair, acts in vitro on a vector into which a target DNA has been inserted and a mutation-introducing DNA to cause homologous recombination. And introduce a site-specific mutation. After that, it may be introduced into a host cell to replicate the plasmid, and the plasmid having the mutation introduced therein may be selected.

The method of the present invention can be carried out more simply by putting together the host cells to be used into a kit. The kit may contain at least a host cell in which a DNA repair system is induced, but may also contain a vector and the like.

[0016]

The present invention will be described in more detail below with reference to examples, but these examples do not limit the present invention in any way.

Example 1 Site-directed Mutagenesis Using Oligonucleotides (1) Construction of pUC19-Mut As a plasmid used in the following examples, downstream 3 of the polycloning site in the lacZ 'gene of pUC19 was used.
A plasmid pUC19-Mu in which 0th G is converted to A
constructed t. The lacZ ′ gene encodes a lacZα-peptide, and when this gene is introduced into a host strain having a genotype of lacZΔM15, β-galactosidase activity is produced. Therefore, in the lacZ ′ gene with no base substitution, growing colonies turn blue due to the reaction using X-gal as a substrate in the presence of IPTG. vice versa,
The base-substituted mutant lacZ ′ gene cannot produce the original activity, and the growing colony becomes white. Utilizing this fact, a mutant lacZ ′ of pUC19-Mut
The efficiency when the base substitution of a gene is restored by site-directed mutagenesis can be calculated from the number of blue colonies and white colonies.

(2) Synthesis of oligonucleotide as mutation-introducing DNA As the mutation-introducing DNA for restoring the base substitution site in the lacZ 'gene, the 5'-end phosphorylated oligo represented by SEQ ID NO: 1 in the Sequence Listing is used. The nucleotide (dR2) was synthesized. That is, dR2 was designed so that the base substitution was restored by the 12th C to become the active lacZ ′ gene.

(3) Preparation of host Escherichia coli A host for electroporation (Escherichia coli BMH7) in which a DNA repair system was induced by the treatment shown in Table 1 below.
1-18mutS strain) was prepared.

[0020]

[Table 1]                             Table 1 ───────────────────────────────────                 UV irradiation Ethyl methanesulfonate Nalidixic acid                     (Sec) (%) (mM)   Host No. 1 30 0.0 0.0   Host No. 2 30 0.1 0.0   Host No. 30 0.1 0.0   Host No. 4 0 0.2 0.0   Host No. 5 0.0 0.0 100   Host No. 6 0 0.0 200   Host No. 7 0 0.0 0.0 ───────────────────────────────────

The host Escherichia coli in which the DNA repair system was induced was prepared as follows. E. coli BMH71-18m
The utS strain (Takara Shuzo) was cultivated in 3 ml of L-medium for 6 hours, diluted 10 ³ times, and spread on LB agar medium. Then, it was irradiated with ultraviolet rays for 30 seconds at a distance of 50 cm with a Hitachi sterilization lamp GL15 (Hitachi), incubated at 37 ° C. in the dark overnight, and the colonies grown there were inoculated into 100 ml of L-medium. And absorbance (660n
Culturing was carried out until about 0.8 in m). When the chemical treatment was carried out with ethyl methanesulfonate and nalidixic acid, they were added so as to reach the respective concentrations when the absorbance reached around 0.4 during the culture. 4,000 the culture
Collect the cells by centrifugation at rpm for 8 minutes,
The cells were suspended in 10 μl of 10% glycerin and used as a host for electroporation.

(4) Introduction into host E. coli The introduction into host E. coli by electroporation is as follows.
It carried out as follows using Gene Pulser (Bio-Rad). For 50 μl of the host prepared in (3),
1 μl of a solution prepared so that dR1 had a concentration of 100 pmol / μl and 0.1 ng of pUC19-Mut were added. This was previously chilled in ice.
It was injected into a dedicated cuvette with a width of 1 cm and a pulse of 1.5 kV was applied. Immediately add 1 ml of SOC medium to 3
The culture was carried out at 7 ° C for 1 hour with shaking. After culturing, the cells were collected, and the whole cells were applied to an LB agar medium containing 100 μg / ml of ampicillin and X-gal and incubated overnight at 37 ° C. After that, the number of grown colonies was counted and the efficiency of site-specific mutation was calculated. When the mutated portion in the lacZ 'gene is restored, the colony becomes blue, and otherwise the colony becomes white. The results are shown in Table 2 below.

[0023]

[Table 2]                               Table 2 ───────────────────────────────────                     Blue-white total (blue / total) x 100   Host No. 1 1 5 6 17   Host No. 2 1 1 2 50   Host No. 3 1 15 16 6.3   Host No. 4 2 7 9 22   Host No. 5 1 3 4 25   Host No. 6 1 1 2 50   Host No. 7 0 23 23 0 ───────────────────────────────────

That is, No. Site-directed mutants were obtained with 50% efficiency when using 2 or 6 hosts.

(5) Analysis of mutation site The blue colony obtained in (4) was treated with ampicillin for 3 m.
The cells were inoculated into 1 L-medium and incubated at 37 ° C. overnight to extract the plasmid. E. coli JM109 was transformed with this and ampicillin and X-g were used.
As a result of application to an L-agar medium containing al, all the grown colonies were blue. As a result of further sequencing and analyzing the mutation site bases, only the restored G was observed, and A was not observed at all. This indicates that even in the same bacterial cell, a plasmid having a mutation introduced thereinto and a plasmid having no mutation introduced therein do not coexist, and only the chain introduced with the mutation is preferentially replicated. As described above, the site-specific introduction, which required at least 3 days in the conventional gapped duplex method or Kunkel method, could be performed in only 1 day.

Example 2 In the case of homologous recombination between site-directed mutagenesis molecules using PCR products, the longer both homologous sequence regions, the more reliable recombination is possible. This example shows that site-specific mutagenesis can be performed with higher efficiency by using a long-chain mutation-introducing DNA obtained by PCR.

(1) Preparation of DNA for mutation introduction by PCR Oligonucleotide (dR1) containing the desired mutation shown in SEQ ID NO: 2 of the Sequence Listing and oligo complementary to another position of the template shown in SEQ ID NO: 3 Using nucleotide (dR3) as a primer pair, pUC19-Mu of Example 1- (1) was prepared.
PCR was performed as follows using t as a template. The composition of the reaction solution was pUC19-Mut 10 ng, primer dR1
And dR3 20 pmol each, dATP, dGTP, d
CTP, TTP 200μM each, Amplitac DNA
Polymerase (Takara Shuzo) 2.5 units, Tris-HC
l (pH 8.4) 10 mM, KCl 50 mM, Mg
Cl ₂ 2.5 mM, gelatin 0.02%, total 1
00μl system, after overlaying mineral oil, 94 ℃ 30
Seconds, 60 ° C. for 30 seconds, 72 ° C. for 1 minute, and 30 cycles were performed. After the reaction, the layered mineral oil was removed, 10 μl of the reaction solution was taken, and the DN of the desired size was electrophoresed.
It was confirmed that the A fragment was amplified. Then, the reaction solution was concentrated and purified to about 10 μl using Microcon 100 (Takara Shuzo).

(2) Introduction into E. coli host 1 μl of the PCR product (156 bp) prepared in (1) and p
UC19-Mut 0.1 ng was added to 50 μl of E. coli B
The mixture was mixed with MH71-18mutS and introduced by ectroporation in the same manner as in Example 1- (4), and the efficiency of site-specific mutation was quantified from the number of colonies. BMH
71-18 mutS is the host No. prepared in Example 1- (3). 2, No. 6 and No. 7 is used, and the conditions for ectroporation are 2.25kV, 200Ω, 2
5μFD, 1.5kV, 100Ω, 25μFD 2
Set to type. The results are shown in Table 3.

[0029]

[Table 3]                             Table 3 ───────────────────────────────────                         Setting conditions Setting conditions   Host No. 2 73/104 (70%) 1/3 (33%)   Host No. 6 86/141 (61%) 2/4 (50%)   Host No. 7 2/124 (2%) 0/7 (0%)                       (Shown as number of blue colonies / total number of colonies) ───────────────────────────────────

In Example 1, a short-chain mutation-introducing DN
When A is used, the mutation efficiency is very sensitive to the conditions of ectroporation, and depending on the set conditions, the mutation efficiency drops to about 10%. However, by using the long-chain mutation-introducing DNA by PCR, a relatively stable mutation efficiency was obtained regardless of the conditions as described above, and the value was successfully increased to 70%. From this fact, using a technique such as PCR and using a long-chain mutation-introducing DNA in the method of the present invention is an effective means for performing site-specific mutation more reliably.

Example 3 Construction of kit for site-directed mutagenesis A kit for site-directed mutagenesis (10 doses) was constructed by setting host Escherichia coli, a control vector and an oligonucleotide (Table 4). In addition, the host Escherichia coli (BMH71-18mutS) was the host No. 1 prepared for eclectoporation in Example 1- (3). 2 was used.

[0032]

[Table 4]                               Table 4 ───────────────────────────────────   BMH71-18mutS (10% glycerin solution) 500 μl   pUC19-Mut (10 ng / μl) 10 μl   dR1 (100 pmol / μl) 10 μl ───────────────────────────────────

[0033]

INDUSTRIAL APPLICABILITY The present invention provides a more convenient site-directed mutagenesis method and a kit for use in the method.

[0034]

[Sequence list]

SEQ ID NO: 1 Sequence length: 22 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: CAGGGTTTTC CCAGTCACGA CG 22

SEQ ID NO: 2 Sequence length: 31 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: GGGTAACGCC AGGGTTTTCC CAGTCACGAC G 31

SEQ ID NO: 3 Sequence length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: GAGCGGATAA CAATTTCACA CAGG 24

Front page continuation (72) Inventor Akihiro Noda 3-4-1 Seta, Otsu City, Shiga Prefecture, Central Research Laboratory, Inc. (72) Inventor Kazuo Nakajima 3-4-1 Seta, Otsu City, Shiga Prefecture Central Research Laboratory (72) Inventor Ikuno Kato 3-4-1 Seta, Otsu City, Shiga Prefecture Sake Brewing Central Research Laboratory (56) References Nucleic Acids Res. , Vol. 21, No. 17 (1993) p. 3977-3980 (58) Fields investigated (Int. Cl. ⁷ , DB name) CA (STN) BIOSIS / WPI (DIALOG) PubMed

Claims (2)

(57) [Claims] 1. A site-directed mutagenesis method comprising introducing a mutation in Escherichia coli in which a DNA repair system is induced , which comprises the following steps: (1) UV irradiation, an alkylating agent, or DNA replication inhibition
Treating the host E. coli by agent, (2) (1) site-specific mutations guide host E. coli cells prepared in
Vector for insertion of target DNA and for mutation introduction
A step of introducing DNA, a step of culturing the transformant prepared in (3) and (2),
A site-directed mutagenesis method including. 2. A kit for use in the site-directed mutagenesis method according to claim 1, which comprises ultraviolet irradiation and alkylation.
A kit for site-directed mutagenesis, which comprises Escherichia coli treated with an agent or a DNA replication inhibitor .