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Site-directed mutagenesis in circular methylated DNA

A technology of methylation and methylase, applied in the field of molecular biology

Inactive Publication Date: 2009-12-09
乔苗
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Various types of site-directed mutagenesis methods currently in use require more additional steps to complete the screening process for mutant DNA, and therefore require more manpower, material resources and time

Method used

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  • Site-directed mutagenesis in circular methylated DNA
  • Site-directed mutagenesis in circular methylated DNA
  • Site-directed mutagenesis in circular methylated DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Partially complementary primers are used in the method for Fip2 site-directed mutagenesis, wherein the mutagenesis site is located in the non-complementary region of one of the primers, and the method steps are as follows:

[0064] 1) Synthesize two partially complementary primers at the 5' end, wherein the mutation is in the non-complementary region, for the introduction of 3 nucleotide substitutions, thereby generating an EcoRV restriction site (capital letters represent 3 cores Nucleotide substitution mutations, bold represents the complementary regions of the forward and reverse primers):

[0065] Primer 1: GaTAtccatcagag-3′, and

[0066] Primer 2: cctgacacttttc-3′;

[0067] 2) Prepare the reaction solution:

[0068] 2.5 μl 10x reaction buffer (BD Biosciences)

[0069] 10ng Fip2 plasmid (GM Biosciences, Inc)

[0070] 0.5 μl Primer 1 (20 μM)

[0071] 0.5 μl Primer 2 (20 μM)

[0072] 1 μl of 10 mM dNTPs (2.5 mM of each dNTP) (BD Biosciences)

[0073] Add do...

Embodiment 2

[0085] Partially complementary mutagenesis primers are used in the method for site-directed mutagenesis of Fip2, and the mutagenesis site is located in the complementary region of the two primers. The steps of the method are as follows:

[0086] 1) Synthesize two partially complementary primers at the 5' end, wherein the mutation is used to introduce a substitution of 3 nucleotides in the complementary region, thereby generating an EcoRV restriction site (capital letters represent 3 nucleotides Substitution mutations, bold represents the complementary regions of the forward and reverse primers) Substitution substitutions:

[0087] Primer 3: ttgaatgaaaag-3′, and

[0088] Primer 4: tttcaagggc-3';

[0089] 2) Prepare the reaction solution:

[0090] 2.5 μl 10x reaction buffer (BD Biosciences)

[0091] 10ng Fip2 plasmid (GM Biosciences, Inc)

[0092] 0.5 μl Primer 1 (20 μM)

[0093] 0.5 μl Primer 2 (20 μM)

[0094] 1 μl of 10 mM dNTPs (2.5 mM of each dNTP) (BD Biosciences...

Embodiment 3

[0107] According to the method steps of the present invention, Fip2 is mutagenized with two completely complementary primers, and the steps are as follows:

[0108] 1) Synthesize two fully complementary primers, where the mutation is used to introduce a 1 nucleotide substitution (capital letters):

[0109] Primer 5: 5′-gagctcctgaccgCgaaccaccagctgaaag-3′, and

[0110] Primer 6: 5′-ctttcagctggtggttcGcggtcaggagctc-3′;

[0111] 2) Prepare the reaction solution:

[0112] 2.5 μl 10x reaction buffer (BD Biosciences)

[0113] 10ng Fip2 plasmid (GM Biosciences, Inc)

[0114] 0.5 μl Primer 1 (20 μM)

[0115] 0.5 μl Primer 2 (20 μM)

[0116] 1 μl of 10 mM dNTPs (2.5 mM of each dNTP) (BD Biosciences)

[0117] Add double distilled water to make the final reaction volume reach 25 μl;

[0118] 3) Incubate the reaction solution prepared in step 2) at 98° C. in a PCR instrument (PTC-200thermocycler, Bio-Rad,) for 30 minutes, and incubate at 95° C. for 5 minutes to denature the template....

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Abstract

The invention provides a new method for conducting site-specific mutation in methylated circular stranded DNA molecules conferred by means of mutagenic primer pairs and methylase deficient Escherichia coli. The mutagenic primer pairs are complementary at 5' end or 3' end, or completely complementary to each other. Firstly, mutagenic primer pair is annealed to opposite strands of the methylated circular double-stranded parent DNA molecules. Then, polymerase chain reaction with DNA polymerase is performed by using unmethylated dNTPs to create unmethylated mutagenized double-stranded daughter DNA molecules. Finally, the reaction mixture of the methylated parent DNA molecules and unmethylated mutagenized daughter DNA molecules is transformed into a methylase deficient competent E.coli. The replication of methylated parent DNA is inhibited in methylase deficient host cell. In contrast, the unmethylated daughter DNA, which contains the desired mutation, are efficiently replicated in methylase deficient host cell and recovered thereafter. The invention also provides a kit for introducing site-specific mutagenesis in accordance with the method of the present invention.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to a new method for site-directed mutagenesis. Background technique [0002] Site-directed mutagenesis is a powerful tool in the field of studying the effects of DNA sequence changes on protein function. Site-directed mutagenesis can be performed in a variety of ways (see, eg, US Patent Nos. 6,391,548, 7,132,265 and 6,713,285, incorporated herein by reference). Polymerase chain reaction (PCR) has been widely used in this field as a cyclic amplification technique in which mutagenic primers are used to introduce desired mutations (see Allemandou et al., J Biomed Biotechnol S, 202-207, (2003); An et al., Appl Microbiol Biotechnol S 68, 774-778, (2005); Jeltsch et al., Methods Mol Biol S 182, 85-94 (2002); Hall, et al. Protein Eng. 4:601 (1991) ; Hemsley, et al. Nucleic Acids Research 17: 6545-6551 (1989); Ho, et al. Gene 77: 51-59 (1989); Hultman, et al. Nucleic Acids Rese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12P19/34C12Q1/68
CPCC12N15/102
Inventor 乔苗
Owner 乔苗
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