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Method for room temperature amplification of DNA

A room temperature amplification and room temperature technology, which is applied in the field of DNA amplification, can solve problems such as interference with detection effects, easy side reactions, and complex reaction systems, and achieve the effects of shortening reaction time, improving speed and efficiency, and simplifying the extension process

Active Publication Date: 2014-09-24
胡振新
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Room temperature PCR relies on recombinases and polymerases with strand displacement to amplify relatively short sequences, and requires an ATP regeneration system, which makes the reaction system more complicated, easily causes side reactions, and interferes with the detection effect

Method used

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  • Method for room temperature amplification of DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 Conventional PCR and comparative experiment of the present invention

[0052] 1. Preparation of template DNA

[0053] The DNA of the pUC18-Rad18B plasmid was extracted using a commercial nucleic acid extraction kit (Qiagen Company) according to its instructions. Wherein, the pUC18 plasmid contains human Rad18B gene.

[0054] Among them, the construction of the pUC18-Rad18B plasmid is as follows:

[0055] Using human cDNA as a template (purchased from Shanghai Tianyu Biotechnology Co., Ltd.), and using AAA TCT AGA TAT GAA TCA TTCCAG CTT TG (shown in SEQ ID NO.1) and AAA GGT ACC TGT CGA CCC CGG CCC GTA G (SEQ ID NO.2) are the upstream and downstream primers, amplified by PCR to obtain the amplified fragment of the human Rad18B gene, and then, the amplified fragment of the human Rad18B gene is digested with XbaI and KpnI to obtain the DNA fragment of the human Rad18B gene , and then, insert the DNA fragment into the pUC18 plasmid (purchased from Shanghai Ti...

Embodiment 2

[0084] Example 2 Room temperature amplification of human gene Rad18B

[0085] 1. Preparation of template DNA

[0086] A commercial nucleic acid extraction kit (Qiagen Company) was used to extract the DNA of the pUC18-Rad18B plasmid according to its instructions to obtain template DNA.

[0087] Wherein, the template DNA is incubated at 30° C. and diluted sequentially by 10 times to obtain diluted template DNA.

[0088] 2. DNA amplification at room temperature

[0089] Mix template DNA (including diluted template DNA) with amplification reagents, and perform DNA amplification at 30°C for 30 minutes (the total reaction volume of DNA amplification is 10 μL) to obtain the DNA amplification product of human Rad18B gene; , the amplification reagent is composed of the following components:

[0090] 1) 20mM pH7.5 Tris-HAc

[0091] 2) 50mM KAc

[0092] 3) 3mM Mg(Ac) 2

[0093] 4) 2mM mercaptoethanol

[0094] 5) Polyethylene glycol (PEG) 20000 with a mass percentage of 2%

[009...

Embodiment 3

[0105] Example 3 Room temperature amplification of human gene Rad18B

[0106] Operate according to the method of Example 2, wherein the amplification reagent is modified to consist of the following components:

[0107] 1) 30mM pH7.8 Tris-HAc

[0108] 2) 80mM KAc

[0109] 3) 5mM Mg(Ac) 2

[0110] 4) 4mM mercaptoethanol

[0111] 5) Polyethylene glycol 20000 with a mass percentage of 3.5%

[0112] 6) 80 μM dNTPs

[0113] 7) 500nM of the forward primer shown in SEQ ID NO.3, 500nM of the reverse primer shown in SEQ ID NO.4

[0114] 8) 0.8mM ATP

[0115] 11) 80nM human polη polymerase

[0116] 12) 1.5 μM human RPA

[0117] 13) 1 μMT4 bacteriophage UV-sensitive enzyme 1

[0118] 14) 1 μM MT4 phage UV-sensitive enzyme 2.

[0119] The DNA amplification products in this example were subjected to polyacrylamide gel (10% non-denaturing) electrophoresis, and the results were as follows: image 3 As shown, the amplified product is only a 226bp product, and there is no detectable...

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Abstract

The invention discloses a method for room temperature amplification of DNA. The method comprises the following steps: (1) extracting template DNA; and (2) mixing the template DNA with an amplification reagent to form an amplification mixed liquor and then carrying out room temperature DNA amplification for 10 to 60 min so as to obtain desired amplified DNA. The method provided by the invention realizes amplification of DNA at normal temperature, makes DNA amplification possible under the condition of limited resources, shortens reaction time, prepares the specific amplification product and simplifies pairing of DNA primers and extension of a DNA polymerase compound.

Description

technical field [0001] The invention relates to a method for amplifying DNA, in particular to a method for amplifying DNA at room temperature. Background technique [0002] Currently, DNA amplification methods include conventional PCR, isothermal PCR, etc. These technologies can rapidly amplify the target DNA by 1 million times or even higher in a short period of time. Since its launch in 1983, conventional PCR has relied on three-temperature (melting, annealing, and extension) cycles, which have very high requirements for the stability of PCR instruments. Although the two-temperature cycle can shorten the reaction time, the DNA polymerase activity is not at the optimal temperature, so only shorter fragments can be amplified, and the time consumption is not greatly reduced. Isothermal PCR relies on a stable temperature above normal temperature (eg 60°C), and still requires a precise temperature-controlled instrument. [0003] Room temperature PCR relies on recombinase and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 胡振新
Owner 胡振新
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