Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
2001-08-02
2004-01-06
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C435S006120, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06673610
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an entirely new method for mutagenesis, which can be applied for introducing certain mutations at certain sites of a nucleotide sequence, or for introducing random mutations at certain sites of a nucleotide sequence.
BACKGROUND OF THE INVENTION
The method for site-directed mutagenesis is a powerful tool for producing mutants to assess the importance of specific amino acid residues in a protein's structure and function. As such method for site-directed mutagenesis, a variety of protocols have been established, including those using the polymerase chain reaction (PCR).
Among the PCR-based protocols, the Quik Change™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA) (referred to as “Quik Change method”, hereinafter) is widely used. According to the protocol of said Quik Change method, a DNA containing mutations can be obtained through the following three steps of:
1) carrying out the PCR process by use of a pair of complementary primers designed to carry a mutation at a desired site;
2) digesting only the template DNAs (that carry no mutations) in the reaction solution; and
3) performing transformation by use of the DNA fragments which are not digested in the above step of digestion.
In particular, said Quik Change method utilizes, in the step of carrying out the PCR process, a supercoiled, double-stranded (ds) DNA plasmid as a template DNA, and a high-fidelity Pfu Turbo™ DNA polymerase as an enzyme. During the step of the PCR process, the pair of complementary primers is annealed to the dsDNA, followed by an elongation reaction so as to amplify DNA fragments that carry mutations and are nicked between the elongated terminus and the 5′-terminus of the primers.
Subsequently, in the Quik Change method, DpnI endonuclease is caused to act in the reaction solution to specifically cut fully- or hemi-methylated 5′-GATC-3′ sequences. The in vitro synthesized and nicked plasmid DNA including the desired mutation is then transformed into
E coli
As a result, the plasmid DNA that is nicked will be repaired in
E coli
and become circular.
Thus, according to the Quik Change method, it is possible to synthesize a DNA including a desired mutation at a desired site. However, the Quik Change method suffers from the following disadvantages:
1) multiple mutations can not be introduced at different relatively separated positions in a nucleotide sequence at the same time, i.e., in this method, it is possible to introduce multiple mutations only in a pair of complementary primers, whereas it is impossible to introduce multiple mutations at remote positions;
2) when introducing a mutation, a pair of synthetic complementary oligonucleotides primers is required, that is, this method requires two primers for introducing a mutation at one site; and
3) since it requires a pair of synthetic complementary oligonucleotides primers, it does not allow random mutagenesis at desired sites using degenerative primers.
The Quik Change method, due to the above problem 1), requires that the aforementioned 3 steps be repeated the number of times corresponding to the number of mutagenesis, when introducing mutations at multiple sites. Consequently, the Quik Change method takes a long time to introduce mutations at multiple sites, leading to an inconvenience of poor efficiency. Additionally, the Quik Change method, owing to the above problem 2), involves high cost in synthesizing or purchasing the pair of synthetic oligonucleotides primers. Moreover, the Quik Change method, due to the above problem 3), can not be used in experimental systems where certain amino acid residues in the analyte protein are mutated into a variety of other amino acids, thus limiting the experimental systems to which it is applicable.
SUMMARY OF THE INVENTION
Thus, in view of the problems of prior art mentioned above, an object of the present invention is to provide an entirely new method for mutagenesis, which method being simple, speedy, economical, and widely-applicable.
The method for mutagenesis according to the present invention, having achieved said object, comprises the following steps of:
a DNA synthesis in which one or more primers which have a nucleotide sequence containing at least one mutation and a phosphorylated 5′-terminus are annealed to a template DNA and then subjected to an elongation reaction using a thermostable high-fidelity DNA polymerase, after which the phosphorylated 5′-terminus and the elongated terminus are ligated by means of a thermostable DNA ligase to synthesize a circular DNA containing said primers;
a digestion in which said step of DNA synthesis is repeated several times to amplify the DNA containing said primers, after which at least the DNAs other than the amplified circular DNA are then digested into several fragments; and
a double-stranded DNA synthesis in which, with the several fragments obtained in the above step of digestion as megaprimers, said megaprimers are annealed to said circular DNA synthesized in the above step of DNA synthesis, followed by an elongation reaction performed using said thermostable high-fidelity DNA polymerase.
REFERENCES:
patent: 5354670 (1994-10-01), Nickoloff et al.
patent: 5605793 (1997-02-01), Stemmer
patent: 5789166 (1998-08-01), Bauer et al.
patent: 5811238 (1998-09-01), Stemmer et al.
patent: 5932419 (1999-08-01), Bauer et al.
patent: 5939250 (1999-08-01), Short
Berger et al. Phoenix Mutagenesis : One-step reassembly of multiply cleaved plasmids with mixtures of mutant and wild-type fragments. Analytical Biochemistry 214 : 571-579 (1993).*
Hermes et al. A reliable method for random mutagenesis: the generation of mutant libraries using spiked oligodeoxyribonucleotide primers. Gene 84 : 143-151 (1989).*
Derbyshire et al. A simple and efficient procedure for saturation mutagenesis using mixed oligodeoxynucleotides Gene 46 : 145-152 (1986).*
Caldwell et al. Randomization of genes by PCR mutagenesis. PCR Methods and Applications 2 : 28-33 (1992).*
Wells et al. Cassette mutagenesis : an efficient method for generation of multiple mutations at defined sites. Gene 34 : 315-323 (1985).*
Smith M. In vitro mutagenesis. Ann. Rev. Genet. 19 : 423-462 (1985).*
Perlak F. Single step large scale site-directed in vitro mutagenesis using multiple oligonucleotides. Nucleic Acids Research 18 (24) : 7457-7458 (1990).*
Liao et al. A simple high-efficiency method for random murtagenesis of cloned genes using forced nucleotide misincorporation. Gene 88 : 107-111 (1990).*
Sarkar et al. The “Megaprimer” method of site-directed mutagenesis. BioTechniques 8 (4) :; 404-407 (1990).*
Picard et al. A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase. Nucleic Acids Research 22(13) : 2587-2591 (1994).*
Ito et al. A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction. Gene 102 (1) 67-70 (1991).*
Landt et al. A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene 96 : 25-128 (1990).*
Ge et al. Simultaneous Introduction of multiple mutations using overlap extension PCR. BioTechniques 22 (1) : 28-30 (1997).
Amplification of closed circular DNA in vitro, Nucleic Acids Research, 1998, vol. 26, No. 4, Pages 1126-1127.
European Search Report.
XP-002179561; Approaches to DNA Mutagenesis: An Overview; Analytical Biochemistry, 254, Pgs. 157-178 (1997); Article No. aB9724B28.
XP-001019207; Two-Stage PCR Protocol Allowing Introduction of Multiple Mutations, Deletions and Insertions Using Quick-Change Site-Directed Mutagenesis; vol. 26, No. 4 (1999).
XP-002096735; An improved PCR-mutagenesis strategy for two-site mutagensis or sequence swapping between related genes; Nucleic Acids Research, 1998, vol. 26, No. 7 Pgs. 1848-1850.
XP-002179562; Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagensis; Nucleic Acids Research, 2000, vol. 28, No. 16.
XP-002179563; Circularly permuted green fluorescent proteins engineered t
Miyawaki Atsushi
Sawano Asako
Riken
Whisenant Ethan
LandOfFree
Method for mutagenesis does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for mutagenesis, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for mutagenesis will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3222081