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Method of point mutation without need of chain reaction of polymerase and the kit

A technology of chain reaction and polymerase, which is applied in the direction of mutant preparation, biochemical equipment and methods, and microbial measurement/inspection. It can solve the problems of time-consuming operation and cumbersome steps, and achieve the effect of low price and simple operation.

Inactive Publication Date: 2005-12-28
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of the present invention can overcome the defects of cumbersome steps and time-consuming operation in the existing point mutation method; making this method into a kit will greatly facilitate users and provide a good technical platform for the research of gene function, protein structure and function

Method used

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  • Method of point mutation without need of chain reaction of polymerase and the kit
  • Method of point mutation without need of chain reaction of polymerase and the kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The molar ratio of primer (phosphorylated at the 5' end) to template is 100:1

[0042] 1. The first step is template denaturation and primer annealing to template

[0043] Template 500ng

[0044] Mutation primer 1 130ng

[0045] Annealing buffer 1 2μl

[0046] H 2 O 20-X

[0047] Reaction conditions: the above mixture was reacted at 100° C. for 5 minutes; rapidly ice bathed for 5 minutes; room temperature for 30 minutes to obtain an annealed mixture.

[0048] X means except H 2 The sum of the volumes of other solutions other than O (the following explanations are the same for X in each embodiment).

[0049] 2. Mutant strand synthesis and nick ligation

[0050] Annealing mixture 20μl

[0051] 10× synthesis buffer 3μl

[0052] T4 DNA polymerase 1μl

[0053] T4 DNA ligase 1 μl

[0054] 10mM dNTPs 1.5μl

[0055] H 2 O 3.5 μl

[0056] Ice bath for 5 minutes, room temperature for 5 minutes, 37°C, water bath for 2 hours.

[0057] 3. The second step of dena...

Embodiment 2

[0076] The molar ratio of the two point mutation primers (phosphorylated at the 5' end) to the template is 200:1

[0077] 1. The first step is template denaturation and primer annealing to template

[0078] Template 500ng

[0079] Mutation Primer 1 260ng

[0080] Mutation Primer 2 260ng

[0081] Annealing buffer 2 2 μl

[0082] H 2 O 20-X

[0083] 100°C for 5 minutes; rapid ice bath for 5 minutes; room temperature for 30 minutes to obtain an annealing mixture.

[0084] Step 2, 3, 4, 5, 6 and 7 are the same as in Example 1.

Embodiment 3

[0086] The molar ratio of primer (phosphorylated at the 5' end) to template is 100:1

[0087] 1. The first step template denaturation and primer annealing template

[0088] Template 500ng

[0089] Mutation primer 1 130ng

[0090] Annealing buffer 1 2μl

[0091] H 2 O 20-X

[0092] 100°C for 5 minutes; rapid ice bath for 5 minutes; room temperature for 30 minutes to obtain an annealing mixture.

[0093] 2. DNA Polymerization and Nick Ligation

[0094] Annealing mixture 20μl

[0095] 10× synthesis buffer 3 μl

[0096] T7 DNA polymerase 1μl

[0097] T4 DNA ligase 1 μl

[0098] 10mM dNTPs 1.5μl

[0099] H2 O 3.5 μl

[0100] 5 minutes in ice bath, 5 minutes at room temperature, 37°C, 30 minutes in water bath.

[0101] 3. The second step of denaturation and annealing

[0102] Add 100ng of selection primers, 100°C for 5 minutes; quickly ice bath for 5 minutes; room temperature for 30 minutes, to obtain a denaturation mixture.

[0103] 4. The second step of aggrega...

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Abstract

The invention relates to a point mutation method without polymerase chain reaction and its kit. In this method, the plasmid template is first denatured, and the primer containing the point mutation is annealed to the template; then DNA polymerase and DNA ligase are added to synthesize the mutant chain and the nick is closed; then the second step of denaturation is performed, and the selection primer is added and combined with the mutation Chain annealing; use polymerase to polymerize to synthesize another mutant chain; finally add DpnI restriction endonuclease to the product to digest and remove the non-mutated template. Since the method does not require PCR, the mutation process is high-fidelity and does not introduce other additional mutations; and the method is cheap, time-saving, fast and easy to operate.

Description

technical field [0001] The invention relates to a point mutation method without polymerase chain reaction and its kit. technical background [0002] With the advent of the post-genome era, the study of gene function has become a hot spot in life science research. Gene site-directed mutagenesis technology has become a basic means and powerful tool for studying gene function, gene modification and vector modification. It has a wide range of potential applications, such as studying the structure of protein interaction sites, modifying different activities or kinetic properties of enzymes, and drug development, gene therapy, etc. Gene site-directed mutagenesis technology initially uses single-stranded DNA as a template, but the preparation of single-stranded DNA is time-consuming and technically difficult, and then this technology has been continuously improved, and has been developed to be able to operate on double-stranded plasmid DNA molecules, and has formed many product. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/01C12P19/34C12Q1/68
Inventor 黄大卫辛文
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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