JP2008109875A - Convenient and sure method for introducing mutation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel method for introducing site-specific mutation which is performed in molecular biological research field. <P>SOLUTION: Disclosed is a method for introducing site-specific mutation into a double-stranded cyclic DNA molecule, wherein the method comprises: (1) performing PCR by using a DNA polymerase having 3'-5' exonuclease activity using the double-stranded cyclic DNA molecule as a template; (2) selectively decomposing the double-stranded cyclic DNA molecule used as the template; and (3) performing self-cyclization by simultaneously acting a phosphorylase and a binding enzyme for DNA on the PCR amplification products. Also disclosed is a kit therefor. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention provides a simple and reliable method for site-directed mutagenesis in the field of molecular biology.

  Site-directed mutagenesis is a method of introducing mutations such as substitutions, deletions, and insertions into specific positions of DNA, particularly DNA cloned into a plasmid. In today's molecular biology field, proteins It is used as a very useful means in functional analysis of the above and identification of functional sites of functional nucleic acids.

  For example, after substituting an amino acid residue at a specific position of a gene with another amino acid or deleting it, by examining changes in the function of that gene, You can guess if you played a role. In gene expression studies, site-directed mutagenesis is used to examine which bases interact with transcription factors in gene expression control regions such as promoters and enhancers.

As site-specific mutagenesis methods, methods based on various principles have been developed so far (Non-Patent Documents 1 to 3, Patent Document 1). The principle common to these site-directed mutagenesis methods is to first anneal a primer containing a mutation to a plasmid serving as a template, and then synthesize a DNA strand containing the mutation in vitro by acting a DNA polymerase, Finally, the clone containing the mutation is isolated by transforming E. coli.
Kunkel, TA (1985) Proc. Natl. Acad. Sci. USA 82, 488. Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271. WO97 / 20950 Stratagene Weiner MP, et al. (1994) Gene. Dec 30; 151 (1-2): 119-23.

Here, the key points in comparing and evaluating the methods are [1] high efficiency of mutation introduction, [2] types of mutations that can be introduced, [3] absence of mutations other than the intended purpose, [4 ] The simplicity of the method. Here, [1] mutagenesis efficiency is the ratio of clones (mutants) containing mutations in the clones finally obtained by transforming E. coli, and of course, the higher the higher Various methods have been adopted in each method in order to increase the efficiency because the labor for screening the mutants can be saved. For example, in the method of Kunkel et al., By introducing uracil in advance into a template plasmid, the plasmid used as the template is distinguished from the DNA strand containing the mutation synthesized in vitro to improve the efficiency of mutation introduction. Yes. In addition, in the method of Weiner MP et al. And WO02 / 38789, utilizing the fact that the template plasmid prepared in E. coli is methylated, it is decomposed by treating with a restriction enzyme specific for methylated DNA, Increases the efficiency of mutagenesis. In any of these methods, a mutagenesis efficiency of 50 to 90% is obtained by these devices.
WO02 / 38789

  However, when each method is evaluated from the viewpoints of [1] to [4], each method has advantages and disadvantages and is not good.

  For example, in the method of Kunkel et al., As described above, by introducing uracil into a template plasmid in advance, it is distinguished from DNA strands containing mutations synthesized in vitro to improve mutation introduction efficiency. . However, in order to introduce uracil, it is necessary to use special E. coli, and in this method, it is also necessary to prepare single-strand DNA using a plasmid having a replication origin of a single-stranded DNA phage such as M13 phagemid. From the viewpoint of simplicity of the method, it is a rather complicated method.

  The method of Hashimoto-Gotoh et al. Is based on the Oligonucleotide-directed Dual Amber method. In this method, when a plasmid having a double amber mutation on the kanamycin resistance gene is introduced into a suppressor-free (sup0) host such as E. coli MV1184, the transformant may exhibit kanamycin resistance. It uses a property that cannot be done. This eliminates the appearance of clones derived from the template plasmid and increases the efficiency of mutation introduction. However, this method is also a difficult method from the viewpoint of versatility and simplicity because there is a limit to the plasmids that can be used and it is necessary to use a suppressor-free (sup0) E. coli host.

  The method of WO97 / 20950 Stratagene is used in a kit marketed by Stratagene under the product name “QuikChange”. In this method, after synthesizing a DNA strand containing a mutation using two mutation-introducing primers complementary to each other, after decomposing the template plasmid with a restriction enzyme DpnI that specifically acts on methylated DNA, It is used for transformation of E. coli. This method has no limitation on the types of plasmids and E. coli that can be used, and is extremely versatile. Moreover, since only two steps are required in terms of operation, the method is extremely simple. However, this method can provide high mutagenesis efficiency for substitution, deletion, and insertion of several bases, but cannot be applied to deletion or insertion of several tens of bases, or has a problem that the mutagenesis efficiency is extremely lowered. There is.

  The method of Weiner MP et al. Is a method based on a so-called inverse polymerase chain reaction (PCR) method. In this method, first, inverse PCR is performed using a primer containing a mutation to synthesize a DNA strand containing the mutation. Next, self-circularization (self-ligation) of the PCR product is performed with DNA ligase, and finally the template plasmid is digested with the restriction enzyme DpnI to be used for transformation of E. coli. This method is also extremely versatile, with no limitation on the types of plasmids or E. coli that can be used. Further, unlike the method of WO97 / 20950 Stratagene, deletion mutants in which several tens or hundreds of base regions are deleted in addition to substitution, deletion, or insertion of several bases can be prepared. Furthermore, there is a feature that Mutant clone collections in which specific positions are substituted with 20 kinds of amino acids can be produced.

  However, the mutation introduction method based on the inverse PCR method has many advantages, but [1] mutations other than the intended mutation (unintended mutation) due to incorporation of a wrong base by the polymerase during PCR. : 2nd-site mutation) is more likely to be inserted than other methods, [2] the full length cannot be amplified with large plasmids, [3] primer phosphorylation is required, [4] operation is There was a drawback that it was relatively multi-stage and complicated. Therefore, there has been a demand for a simpler method without these disadvantages.

In order to overcome these disadvantages, Non-Patent Document 4 uses Vent DNA polymerase, which is a DNA polymerase having 3′-5 ′ exonuclease activity, which is an enzyme with extremely high fidelity to PCR enzymes. By reducing the insertion of external mutations (2nd-site mutation) and making the PCR product blunt end (blunt ends), a reaction between phosphorylase (polynucleotide kinase) and DNA-binding enzyme (DNA ligase) after PCR We have reported a system in which self-cyclization can be carried out easily. However, in this system, PCR is performed for 25 cycles, so there is still a high possibility that unintended mutations will be introduced. Furthermore, after the PCR reaction, the PCR amplification products containing the mutations are separated by agarose gel electrophoresis and separated from the gel. The target band had to be cut out and purified, which was still a complicated operation. Furthermore, this method was a method requiring two days at the shortest.
Nucleic Acids Res. 1991 May 25; 19 (10): 2785. A simple and rapid method for generating a deletion by PCR. Imai Y, Matsushima Y, Sugimura T, Terada M.)

  In light of the above circumstances, the present inventors have developed a simple mutation introduction method that is extremely unlikely to introduce an unintended mutation during PCR in a mutation introduction method based on the inverse PCR method, as a result of earnest research. As a result, the present invention has been completed.

That is, the present invention relates to (1) a method for introducing a site-specific mutation into a double-stranded circular DNA molecule, [1] 3′-5 ′ exo using a double-stranded circular DNA molecule as a template. PCR using a DNA polymerase having nuclease activity, [2] selective degradation of the double-stranded circular DNA molecule used as the early template, [3] It is a method comprising the steps of causing DNA-binding enzymes to simultaneously act and self-circulate. The present invention also provides (2) a reagent kit for site-specific mutation introduction into a double-stranded circular DNA molecule, [1] a DNA polymerase having 3′-5 ′ exonuclease activity, [2] A reagent kit comprising phosphorylase, [3] DNA-binding enzyme, and [4] restriction enzyme.

  According to the present invention, mutagenesis can be carried out more reliably, quickly and simply than conventional methods while suppressing the possibility of insertion of unintended mutations in the site-directed mutagenesis method based on inverse PCR. Can do.

The present invention will be described in detail below.
The DNA polymerase having 3′-5 ′ exonuclease activity in the present invention is a DNA polymerase having an activity of digesting a DNA strand from the end in the direction from the 3 ′ end to the 5 ′ end of the DNA and cutting it. In general, enzymes used for PCR are roughly classified into two types. One is a DNA polymerase having no 3′-5 ′ exonuclease activity represented by Taq DNA polymerase, which has been used from the beginning of PCR method development, and the other is a 3′-5 ′ exonuclease activity (DNA calibration activity). A DNA polymerase. The former is sometimes called Pol I type DNA polymerase, and the latter is called α type DNA polymerase. Examples of the DNA polymerase having 3′-5 ′ exonuclease activity in the present invention include KOD DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase, Ultima DNA polymerase, etc. that have the activity alone, Taq KOD Dash DNA polymerase, EX-Taq DNA polymerase, Expand DNA polymerase prepared by mixing DNA polymerase having 3'-5 'exonuclease activity with DNA polymerase having no 3'-5' exonuclease activity such as DNA polymerase Etc. are also included. A characteristic of a DNA polymerase having 3′-5 ′ exonuclease activity is its high fidelity. That is, the frequency of incorporating an incorrect base during DNA synthesis is extremely low, and its complementary strand can be synthesized faithfully to the template. Another feature of a DNA polymerase having 3′-5 ′ exonuclease activity is that the ends of DNA fragments amplified using the polymerase become blunt ends. As a result, the end of the DNA fragment can be connected to the other end and self-circularized (self-ligation). In contrast, Pol I-type enzymes such as Taq DNA polymerase have low fidelity and may incorporate wrong bases during the DNA polymerase reaction. Further, since the 3 ′ end of the amplified DNA fragment is a protruding end of dA, self-circulation cannot be performed as it is. The DNA polymerase used in the present invention is not particularly limited as long as it is a high fidelity DNA polymerase having 3′-5 ′ exonuclease activity.

PCR (polymerase chain reaction) is a technique for amplifying a target DNA fragment in a short time using a DNA polymerase, using a template to be amplified and a set of two primers designed to sandwich the region to be amplified. A thermal cycling reaction is performed, and the target DNA is amplified exponentially. In the present invention, among PCR methods, a method called an inverse PCR method is carried out. Inverse PCR is a technique in which PCR is performed using circular DNA (such as a plasmid) as a template and two types of primers set in the opposite direction (outside) to amplify the entire plasmid. However, in the present invention, when it is desired to prepare a deletion mutant, PCR may be carried out by omitting a part of the plasmid rather than the entire periphery of the plasmid.

A phosphorylating enzyme is an enzyme that adds a phosphate group. In the present invention, it refers to a polynucleotide kinase that adds a phosphate group to DNA. The DNA phosphorylated by this enzyme can serve as a substrate for the subsequent DNA-binding enzyme. In the field of molecular biology, T4 polynucleotide kinase derived from T4 phage is generally used. However, in the present invention, any enzyme capable of adding a phosphate group to the 5 ′ end of DNA may be used, and the origin is not particularly limited. .

A DNA binding enzyme is an enzyme that links DNAs. In the field of molecular biology, T4 DNA ligase derived from T4 phage, E. coli DNA ligase derived from E. coli, Pfu DNA ligase derived from Pyrococcus furiosus, etc. are known. However, in the present invention, it is only necessary that the 5 ′ end and 3 ′ end of the 5′-end phosphorylated PCR product can be linked, and the present invention is not particularly limited thereto. Since the PCR amplification product in the present invention has blunt ends (blunt ends), T4 DNA ligase that can efficiently link blunt ends is preferably used.

The self-circulation (self-ligation) is to circularize by connecting the 5 ′ end and the 3 ′ end within the same DNA molecule, not between DNA molecules. In the present invention, this makes the PCR amplification product a circular plasmid, which can be transferred (transformed) into E. coli. Since the plasmid transferred to E. coli forms an E. coli colony on an agar medium containing an appropriate antibiotic, the mutant can be isolated.

The step of selectively degrading DNA derived from a double-stranded circular DNA molecule used as a template is performed for the purpose of degrading a template that does not contain the target mutation and facilitating selection of mutants. is there. As a method of selectively degrading DNA derived from a double-stranded circular DNA molecule used as a template, it is necessary to distinguish the template DNA from the DNA strand synthesized by the PCR reaction with a substantial difference. (1) A method in which uracil (dUTP) is substituted for thymine (dTTP) in the template DNA, and this is treated and decomposed with uracil-DNA glycosylase (UNG). (2) A method in which methylated plasmid DNA is used as a template and treated and decomposed using a methylated DNA-specific restriction enzyme or the like. In the method of (1), uracil (dUTP) is inserted in the template DNA in place of thymine (dTTP). The gene encoding dUTPase (dut) and the gene encoding uracil DNA glycosylase (ung) are deleted. There are a method for preparing DNA in vivo using E. coli strain CJ236, and an in vitro method using uracil (dUTP) instead of thymine (dTTP) in a DNA synthesis reaction such as a PCR reaction. As a method for obtaining methylated plasmid DNA of (2), in addition to the in vivo method of preparing plasmid DNA in general E. coli having methylase, methylase is allowed to act on the isolated plasmid in vitro. The methylated plasmid DNA can also be obtained.

As a method for specifically degrading methylated DNA, a methylated DNA-specific restriction enzyme (restriction endonuclease) is effective, but other enzymes that inactivate methylated DNA may be used. In the present invention, DpnI is suitable as a methylated DNA-specific restriction enzyme, but is not particularly limited as long as it is a methylated DNA-specific restriction enzyme such as NanII, NmuDI and NmuEI, and is an isoschizomer of DpnI. There is no need. In one embodiment of the invention, the template plasmid DNA is dam methylated double stranded DNA and the restriction enzyme used to digest it is DpnI.

In the mutation introduction method based on the inverse PCR method, the number of cycles of the thermal cycling reaction in PCR not only affects the time required for the reaction, but also affects the frequency of introduction of mutations other than the target site (2nd-site mutation). It is a very important factor. That is, in the PCR reaction, even if a high-fidelity DNA polymerase is used, an erroneous base is incorporated as a PCR error with a very low frequency, resulting in a base sequence different from the template. Of course, the possibility that the wrong base is incorporated increases as the polymerase reaction is repeated. Therefore, the smaller the number of cycles in the PCR reaction, the less the possibility of introducing an unintended mutation. However, if the number of cycles is small, sufficient amplification products cannot be obtained, and it is possible that E. coli colonies cannot be obtained even when used for transformation. In the present invention, by optimizing the reaction conditions in each step, the efficiency of the reaction is improved, and E. coli colonies can be obtained even with a small number of PCR cycles. That is. In the present invention, even if the number of cycles is 15 cycles or less, sufficient colonies can be obtained, and therefore the possibility that erroneous bases are incorporated as PCR errors can be minimized.

One of the features of the present invention is that each reaction step can be carried out substantially continuously. That is, in the present invention, the reaction step comprises (1) PCR using a double-stranded circular DNA molecule as a template and DNA polymerase having 3′-5 ′ exonuclease activity, (2) Selectively degrading the double-stranded circular DNA molecule used,
(3) The PCR amplification product is composed of three steps of simultaneously causing a phosphorylase and a DNA-binding enzyme to act on each other to be self-circularized. During these steps, a reagent is simply added, or After transferring a part of the reaction solution to another container, the reagent is simply added. That is, it is possible to proceed the steps without separating and purifying the PCR product by agarose gel electrophoresis or the like, or by performing the buffer replacement by ethanol precipitation or the like. Conventional method (Nucleic Acids Res. 1991 May 25; 19 (10): 2785. A simple and rapid method for generating a deletion by PCR. Imai Y, Matsushima Y, Sugimura T, Terada M.) (Non-patent Document 4) Then, since these operations were essential, not only the reaction step was complicated, but also the reaction time required 2 days. On the other hand, in the present invention, the reaction up to transformation can be completed in about 3 hours.

One embodiment of the present invention is a reagent kit for introducing mutations into a double-stranded circular DNA molecule in a site-specific manner, and includes the following configuration.
(A) a DNA polymerase having 3′-5 ′ exonuclease activity;
(B) Phosphorylase (c) DNA-binding enzyme (d) Restriction enzyme

  The reagent kit of the present invention further includes a reaction buffer for DNA polymerase, if necessary. Further, the phosphorylating enzyme, the DNA binding enzyme, and the restriction enzyme may be contained in separate tubes, or may be configured in a premixed form.

Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1 Evaluation of mutagenesis efficiency when targeting plasmids of various sizes
Attempts to introduce mutations of 1 base substitution, 3 base deletion, 6 base insertion in 5 human cDNA clones (Toyobo FLJ cDNA Clone: Code No. FLJ-101) with a plasmid size of 3.3 kb to 11.3 kb It was. The reaction was performed as follows. That is, according to the instruction manual for KOD-Plus- (Toyobo) having 3'-5 'exonuclease activity, the attached 10x buffer was 1x concentration, the magnesium sulfate solution was 1.2 mM, dNTPs was 0.2 mM, KOD-Plus- A PCR reaction solution was prepared so that 1U (Toyobo), 15 pmol each of the forward PCR primer and reverse primer, 50 ng of the template plasmid, and 50 μl of the final solution volume were prepared. PCR was performed under the following conditions. That is, GeneAmp PCR system 9600 manufactured by Applied Biosystems was used as the reaction equipment, and after heat denaturation reaction 94 ° C · 2 minutes, heat denaturation reaction 98 ° C · 10 seconds, annealing / extension reaction 68 ° C · X min. X cycles were performed. Here, X was set to 1 minute or 1 cycle per 1 kb size of the target plasmid. For example, for a 3.3 kb plasmid, a heat denaturation reaction of 94 ° C. for 2 minutes followed by a heat denaturation reaction of 98 ° C. for 10 seconds, an annealing / extension reaction of 68 ° C. for 3.5 minutes, 11.3 kb For a plasmid having a size of 1, a heat denaturation reaction of 94 ° C. for 2 minutes followed by a heat denaturation reaction of 98 ° C. for 10 seconds and an annealing / extension reaction of 68 ° C. for 11.5 minutes was performed in 12 cycles. Next, in order to digest the template plasmid, 10 units of the restriction enzyme DpnI was added to the reaction solution after completion of the PCR reaction and incubated at 37 ° C. for 1 hour. Next, in order to perform self-ligation of PCR products, 2 μl of DpnI-treated PCR reaction solution, 7 μl of sterile distilled water, 5 μl of Ligation high (Toyobo), 1 μl of T4 Polynucleotide kinase (= 5 units) (final solution volume is 15 μl) Was prepared and stirred well, and then incubated at 16 ° C. for 1 hour. Finally, E. coli was transformed using 10 μl of this reaction solution and 100 μl of E. coli DH5α (Toyobo) according to a conventional method, and an appropriate amount was spread on LB medium (including ampicillin). Next, in order to estimate the mutagenesis efficiency, 12 plasmids were prepared at random from the transformant colonies, sequenced, and the proportion of clones containing the target mutation was evaluated. The results are shown in Table 1. From these results, according to the method of the present invention, the desired mutants could be obtained at an average rate of 84% in the 3.3 kb to 11.3 kb plasmid without being particularly affected by the size.

Example 2 Performance comparison with other methods in large deletion introduction and His-tag insertion
Using the method of the present invention and the method of WO9720950, deletion of 90 bases and introduction of 18 bases corresponding to 6 × His were performed on the 7.3 kb plasmid FLJ32066. The method of the present invention was carried out with the reaction composition and reaction method described in Example 1. On the other hand, for the method of WO9720950, a kit “QuikChange II” commercially available from Stratagene was used according to the instruction manual. As in Example 1, the mutation introduction rate was evaluated by randomly preparing 12 plasmids each from the transformant colonies, sequencing them, and calculating the proportion of clones containing the desired mutation. did. The results are shown in Table 2. From these results, it was found that according to the method of the present invention, mutation that is difficult by other methods such as deletion of 90 bases and introduction of 18 bases corresponding to 6xHis can be carried out with high mutagenesis efficiency. .

Example 3 Evaluation of insertion frequency of unintended mutation (2nd site-mutation) Plasmid pAK119M (mutation was introduced (single base substitution) by the method of the present invention and the method of WO9720950 for the full length. Since pAK119M has a single base substitution, the transformant (colony) becomes blue, so that the ratio of transformants with mutations can be easily evaluated. The PCR was carried out in 8 cycles in the same manner as in Example 1. On the other hand, for the method of WO9720950, a kit “QuikChange II” commercially available from Stratagene was carried out according to the instruction manual. After the mutagenesis operation, transformation was performed, and an appropriate amount was placed in an LB medium (containing ampicillin) containing 0.01% X-gal. Next, in order to evaluate the frequency of insertion of non-target mutations (2nd site-mutation) in each method, 24 clones were selected from the mutants obtained by each method (colony showing blue color). After randomly selecting and preparing the plasmid, the 2000 bp region was sequenced, and as a result, of the 48000 bases (= 24 clones x 2000 bp) actually analyzed according to the present invention, there was only one base mutation other than the intended one. Therefore, it was confirmed that the frequency of introducing unintended mutations was very low, which was equivalent to or better than the method of WO9720950, which is a non-PCR method.

The method of the present invention can introduce mutations very easily and reliably compared to conventional mutation introduction methods. Furthermore, it is possible to create deletion mutants and insertion mutants of several tens of bp, which was difficult with the conventional method. This feature is very useful in protein functional analysis and functional sequence region analysis of DNA, etc., and is economical for conducting mutation introduction regardless of basic research or industrial use research through rapid research and development. Bring great benefits.

It is a figure which shows the flow of the mutation introduction method by this invention.

Claims (6)

A method for introducing a site-specific mutation into a double-stranded circular DNA molecule, comprising the following steps.
(1) PCR is performed using a double-stranded circular DNA molecule as a template and a DNA polymerase having 3′-5 ′ exonuclease activity.
(2) selectively decomposing the double-stranded circular DNA molecule used as the initial template.
(3) A phosphorylase and a DNA-binding enzyme are simultaneously acted on the PCR amplification product to cause self-circulation.   The method according to claim 1, wherein the PCR reaction cycle is 15 cycles or less.   The method according to claim 1, wherein each step is performed continuously.   The method according to claim 1, wherein the means for selectively degrading DNA derived from a double-stranded circular DNA molecule is performed by a restriction enzyme.   The method according to claim 4, wherein the restriction enzyme is DpnI. A reagent kit for site-specific mutation introduction into a double-stranded circular DNA molecule, the reagent kit comprising: (a) a DNA polymerase having 3′-5 ′ exonuclease activity;
(B) Phosphorylase (c) DNA-binding enzyme (d) Restriction enzyme