Constant temperature nucleic acid amplification reaction reagent

A nucleic acid amplification reaction and reagent technology, which is applied in the field of constant temperature nucleic acid amplification reaction reagents, can solve problems such as low yield of amplification products, high requirements for primers, and high purity requirements for amplified template nucleic acids, so as to improve amplification efficiency , low requirements, and high-efficiency amplification effect

Active Publication Date: 2017-09-19
刘琳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there are also some defects in the existing constant temperature amplification technology, such as the low yield of amplified products, high requirements for designed primers, inability to perform multiple amplification and low and poor multiple amplification efficiency, which is not effective for amplifying template nucleic acid. Higher purity requirements and other issues

Method used

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  • Constant temperature nucleic acid amplification reaction reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] The addition of spermidine and formamide in the reaction system compared with no addition

[0091] Reaction system 1 (total volume 50 μL) (containing BstDNA polymerase, spermidine, formamide): the volume percentage content of glacial acetic acid is 2%; magnesium chloride 30 mM; helicase 12 ng / μL; the volume percentage content of formamide 0.2%; spermidine 1ng / μL; betaine 1ng / μL; UvsX protein 45ng / μL; UvsY protein 45ng / μL; single-chain binding protein 700ng / μL; bovine serum albumin 1.5mg / mL; dNTPs 75mM; Tris Base 15mM; pH 7.5.

[0092] Reaction system 2 (total volume 50 μL) (containing BstDNA polymerase, without spermidine and formamide): 2% glacial acetic acid by volume; 30 mM magnesium chloride; 12 ng / μL helicase; 1 ng / μL betaine ; UvsX protein 45ng / μL; UvsY protein 45ng / μL; single-chain binding protein 700ng / μL; bovine serum albumin 1.5mg / mL; dNTPs 75mM; Tris base 15mM;

[0093] Take out 1 reagent tube 1# prefilled with 25 μL reaction system 1; and 1 reagent tube 2#...

Embodiment 2

[0096] The effect of primer design on amplification

[0097] Take out 2 reagent tubes preloaded with 25 μL reaction system one (Example 1) and mark them as 3# and 4# respectively, add 2 μL of F16-1 (20 μM) and R16-1 (20 μM) into the 3# tube, and add Add 2 μL of F16-2 (20 μM) and R16-2 (20 μM) to tube 4#; and add 5 μL of HPV16DNA template DNA I# and 1U of BstDNA polymerase to tubes 3# and 4# respectively, and then Add 13.5 μL of sterilized water to tubes 3# and 4# respectively.

[0098]Mix tubes 3# and 4# respectively, put them into a metal bath or water bath at 37°C, and react for 15 minutes. Then take it out, add 50 μL of 1:1 phenol chloroform reagent, shake and mix well, centrifuge at 12000 for 30 seconds, take 5 μL of supernatant for agarose gel electrophoresis, it can be seen that there is one in the electrophoresis results of tubes 3# and 4# A single band of comparable brightness. see attached results figure 2 .

[0099] Take out 2 reagent tubes preloaded with 25 μL...

Embodiment 3

[0102] Requirements of Spermidine and Formamide on the Purity of Nucleic Acid Templates

[0103] Take out 1 reagent tube 7# preloaded with 25 μL reaction system 1 (Example 1); and 1 reagent tube 8# preloaded with 25 μL reaction system 2 (Example 1), and put them into 7# and 8# tubes respectively Add 2 μL of F16-1 (20 μM) and R16-1 (20 μM); and add 5 μL of HPV16 DNA template DNA II# and 1 U of BstDNA polymerase to tubes 7# and 8# respectively, and then add sterilized water respectively 13.5μL into 7# and 8# tubes.

[0104] Mix tubes 7# and 8# respectively, put them into a metal bath or water bath at 37°C, and react for 15 minutes. Then take it out, add 50 μL of 1:1 phenol chloroform reagent, shake and mix well, centrifuge at 12000 for 30 seconds, take 5 μL of supernatant for agarose gel electrophoresis, it can be seen that there is a single band in the electrophoresis result of No. 7 tube ; while the amplification result of No. 8 tube was diffuse and no bands were seen. see ...

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Abstract

The application relates to a constant temperature nucleic acid amplification reaction reagent. The constant temperature nucleic acid amplification reaction reagent is prepared from helicase, formamide, spermidine, magnesium chloride, glacial acetic acid, UvsX protein, UvsY protein, single strand binding protein, bovine serum albumin, dNTPs, Tris alkali and constant temperature DNA polymerase.

Description

technical field [0001] The invention relates to a constant temperature nucleic acid amplification reaction reagent. Background technique [0002] There are several limitations in the technical application of traditional PCR: first, expensive instruments are required, and at the same time, the instruments need to have sophisticated temperature control procedures and heating templates, so as to realize the denaturation of DNA template strands at very high temperatures. Annealing at low temperature uses primers to extend the template; second, the system needs a high temperature resistant polymerase; third, in the PCR cycle, the annealing time of each cycle is very short, which requires that the primer must quickly find the template In order to achieve extension, this requires PCR to have an excess of PCR primers, and excess primers will mismatch with the template, causing false amplification or primer-dimers. [0003] Compared with the traditional PCR technology, the constant ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844
Inventor 刘琳
Owner 刘琳
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