Method for effecting site-directed mutagenesis

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Reexamination Certificate

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C435S006120, C435S091100, C435S091200, C435S091420, C536S023100, C536S024300, C536S024330

Reexamination Certificate




This application is the national phase under 35 U.S.C. §371 of prior PCT International Application No. PCT/JP97/02355 which has an International filing date of Jul. 7, 1999 which designated the United States of America.
The present invention concerns a method for performing site-directed mutagenesis used in genetic engineering techniques, more easily and efficiently, and a kit for the use in the above method.
In the field of genetic engineering, recently, it is often difficult to obtain a gene product in large amounts while solely relying on techniques of cloning gene to express, and successful cases are few. For this reason, in order to increase a level of expression of a product of a cloned gene, a technique for coinciding a frame and a technique for altering base sequences near the initiation codon without altering amino acid sequences (silent mutation) are minimally required techniques. In addition, in order to carry out cloning of genes and produce more useful protein as a protein to be expressed, a technique is important and essential, which alters the base sequence of a corresponding codon to delete or substitute amino acids and thereby modifies specificities of the protein, such as optimal pH, stability, substrate specificity, Km value, and the like, in the cases where the protein is an enzyme.
As described above, a method for changing a particular base sequence in a cloned gene as desired, i.e. site-directed mutagenesis, is essential to carry out structural and functional analyses of various regulatory regions on genes including RNA, and protein engineering applications using recombinant DNA techniques. In addition, in the research field of protein engineering, a method for performing site-directed mutagenesis is more important, in a view of more rapid and accurate research by introduction of mutations and elimination at the DNA level.
Conventionally, a method for performing site-directed mutagenesis comprises, for instance, the following procedures:
(1) First, a desired DNA to be mutation-introduced is inserted to a vector. Thereafter, the complementary strand of the desired DNA is dissociated by heat-denaturation, in the case of using a double-stranded plasmid DNA, or a M13 phage vector is used to prepare a single-stranded DNA;
(2) An oligonucleotide designed to introduce a desired mutation (mutagenic primer) is annealed with the above single-stranded DNA. Thereafter, a complimentary strand DNA is synthesized in vitro system by a reaction of DNA polymerase and DNA ligase;
Escherichia coli
is transformed with the DNA obtained in the above item (2), and then a clone in which a mutation is introduced is selected.
However, since a ratio of a mutant to a parent DNA is extremely low in the case of carrying out only the above procedures, it is necessary to efficiently select a clone resulting from annealing to a mutagenic primer. Therefore, in the step of the above item (3), there is employed a system for selective removal so that a clone harboring the parent DNA does not grow.
Examples of such system are a method utilizing amber mutation (amber codon) [
Nucleic Acids Research
, 12, No. 24, pp.9441-9456 (1984)]; a method utilizing restriction endonuclease site [
Analytical Biochemistry
, 200, pp.81-88 (1992); Gene, 102, pp.67-70 (1991)]; and a method utilizing dut (dUTPase) mutation and ung (uracil DNA glycosilase) mutation [
Proceedings of the National Academy of Sciences of the USA
, 82, pp.488-492 (1985)], etc. However, in these methods of mutagenesis, their procedures are complicated, and much time is consumed. In addition, in these methods, a proportion of obtaining desired clones in which a mutation is introduced is low.
On the other hand, a method of site-directed mutagenesis described in Japanese Patent Laid-Open No. Hei 7-289262 is a method utilizing amber mutation using DNA polymerase and DNA ligase. As compared to the above methods, although in the method described in Japanese Patent Laid-Open No. Hei 7-289262, mutants can be produced at a high efficiency, the two-step transformation into
Escherichia coli
is necessary, thus hampering simple operation for practically purposes.
In recent years, methods for site-directed mutagenesis based on the wide-spread use of PCR techniques have been developed.
For example, there has been known a method is known, the method comprising the steps of synthesizing a DNA strand to be mutation-introduced using three or more kinds of primers, the primers including a mutagenic primer; thereafter cutting out a DNA strand with restriction endonucleases and then ligating to another vector the resulting DNA strand in which a mutation is expected to be introduced; and transforming a host
Escherichia coli
with the resulting vector. In addition, Quik Change™ Site-Directed Mutagenesis Kit, manufactured by Stratagene, can be used to obtain a mutation-introduced DNA by means of synthesizing a strand with PCR or DNA polymerase from
Pyrococcus furiosus
using two kinds of complementary mutagenic oligonucleotides (mutagenic primers), the oligonucleotides being capable of hybridizing to double-stranded DNA to which mutation is to be introduced, thereafter, digesting with restriction endonuclease DpnI the resulting strand without mutation, and then transforming a host
Escherichia coli
with resultants treated with DpnI. Furthermore, there has been known a method capable of obtaining a mutation-induced DNA comprising adding class IIS restriction endonuclease recognition site at 5′-terminal side of each two kinds of mutagenic oligonucleotides (mutagenic primers), synthesizing strands by PCR, thereafter digesting with class IIS restriction endonuclease a mutation-introduced DNA, ligating and then transforming a host
Escherichia coli
(U.S. Pat. No. 5,512,463).
As described above, the conventional method using DNA polymerase and DNA ligase necessitates such enzyme reactions and a plurality of steps of transformation for fixing mutation sites, consuming too much time, thereby making it difficult to increase efficiency. Also, in the methods utilizing amber mutation or “dut” and “ung” mutations, single-stranded DNA must be isolated. The method for removing restriction endonuclease site has drawbacks, including limited availability of restriction endonucleases.
In view of the above, a method for performing site-directed mutagenesis using the recently widely spread PCR technique has been developed and brought into actual application. However, its operation is complicated by necessitating three or more kinds of primers, including mutagenic primers, the primers including two or more kinds of mutagenic primers, and by necessitating a restriction endonuclease reaction during the operation, and other aspects. There have been other various problems including extremely reduced mutation efficiency that can result from incomplete restriction endonuclease reaction.
Therefore, an object of the invention is to provide a simple and practical method for performing site-directed mutagenesis using PCR method, and a kit for carrying out the method for performing site-directed mutagenesis.
As a result of intensive investigation to develop an efficient and simple method for performing site-directed mutagenesis, the present inventors have succeeded to obtain a clone in which a desired mutation is introduced, only once transforming
Escherichia coli
after carrying out PCR. The present invention has been completed, based on such a fact.
Accordingly, the gist of the present invention follows:
[1] a method for performing site-directed mutagenesis characterized in that the method comprises the step of carrying out PCR by the use of a double-stranded DNA vector having one or more amber codons, the vector resulting from insertion of a target DNA fragment for site-directed mutagenesis, and at least two kinds of selection primers;
[2] the method for performing site-directed mutagenesis according to the above item [1], wherein the selection primers are m


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