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A kind of optimization method of polymerase chain reaction

A technology of chain reaction and optimization method, which is applied in the optimization field of polymerase chain reaction, can solve the problems of complicated extraction and purification of SSB protein, short biological activity retention period, expensive commercial kits, etc., and achieves easy storage and accurate The effect of sample addition, reagent uniformity and specificity improvement

Inactive Publication Date: 2019-02-01
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex technology of extracting and purifying SSB protein and the high requirement of reagent purity, the preparation cost is very high, and the commercial kit is very expensive, and the price is 6-7 times that of conventional PCR reagents; at the same time, in order to maintain the biological activity of the single-chain binding protein, Requires strict storage at -20°C, and its biological activity retention period is short

Method used

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  • A kind of optimization method of polymerase chain reaction
  • A kind of optimization method of polymerase chain reaction
  • A kind of optimization method of polymerase chain reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Add pure PEI to optimize the re-amplification PCR reaction with severe non-specific amplification.

[0058] When carrying out some amplification experiments with few target templates or extremely rare samples, it is often found that the target band cannot be obtained in one amplification. The second amplification increases the yield of the target band in the product. However, while using re-amplification to increase the yield, some non-specific amplification will also be formed.

[0059] In view of such PCR re-amplification that forms non-specific amplification, it is optimized by adding an effective amount of dendrimer to the PCR re-amplification system.

[0060] Specifically, the following steps are included:

[0061] 1. Prepare the PCR system for the first amplification.

[0062] The system composition is as follows:

[0063] Takara rTaq enzyme (5U / μL)

0.125μL

10×PCR buffer (without Mg 2+ )

2.5 μL

dNTP substrate (2.5mM)

2μL

...

Embodiment 2

[0073] Add {(Au) with Au / PEI molar ratio of 100:1 100 -PEI-mPEG 24}Optimize the re-amplification PCR reaction with severe non-specific amplification.

[0074] The reaction system and specific process are the same as in Example 1, but the difference is that the added optimization agent is self-made {(Au) 100 -PEI-mPEG 24}, the concentration is 1.0 μg / μL, and the molecular weight is 92700. Before use, it needs to be diluted 10 times in an ultra-clean bench before use.

[0075] Amplification results such as image 3 shown. From left to right: M: Molecular weight markers (DL2000 from Takara Company, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); 1 is not added {(Au) 100 -PEI-mPEG 24}The re-amplification system of aqueous solution; 100 -PEI-mPEG 24} re-amplification system; 7 is the blank control without adding template. It can be seen that adding {(Au) 100 -PEI-mPEG 24} is 0.3-0.42mg / L, the non-specific amplification in PCR amplification results is significantly reduced, ...

Embodiment 3

[0077] Add {(Au) with Au / PEI molar ratio of 200:1 200 -PEI-mPEG 24}Optimize the re-amplification PCR reaction with severe non-specific amplification.

[0078] The reaction system and specific process are the same as in Example 1, but the difference is that the added optimization agent is self-made {(Au) 200 -PEI-mPEG 24}, the concentration is 1.0 μg / μL, and the molecular weight is 112400. Before use, it needs to be diluted 10 times in an ultra-clean bench before use.

[0079] Amplification results such as Figure 4 shown. From left to right: M: Molecular weight markers (DL2000 from Takara Company, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); 1 is not added {(Au) 200 -PEI-mPEG 24}The re-amplification system of aqueous solution; 200 -PEI-mPEG 24} re-amplification system; 6 is the blank control without adding template. It can be seen that adding {(Au) 200 -PEI-mPEG 24} is 0.32~0.39mg / L, the non-specific amplification in PCR amplification results is significantly reduce...

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Abstract

The invention relates to an optimization method of a polymerase chain reaction. In the optimization method, polyethyleneimine (PEI) or a gold-nanoparticle-entrapped PEI composite material with an effective dosage is added into the polymerase chain reaction (PCR) as an additive. The optimization method has the advantages that an amplification optimization effect is obvious, and the additive is low-cost, is easy to store and is widely applied. The specificity of an amplified product is obviously improved; the yield of products in some reactions is also obviously improved; the optimization method has potential and high application value in the fields of gene detection and cloning, genetic analysis, clinical diagnosis, gene chips and the like.

Description

technical field [0001] The invention belongs to the field of polymerase chain reaction, in particular to an optimization method of polymerase chain reaction. Background technique [0002] Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments, which can be regarded as special DNA replication outside the body. PCR technology was first conceived by Mullis of the United States in 1983. In 1985, he invented the polymerase chain reaction, which is a simple DNA amplification method, which means the real birth of PCR technology and won the 1993 Nobel Prize in Chemistry. In 1973, Taiwanese scientist Qian Jiayun discovered the stable Taq DNA polymerase and made a fundamental contribution to the development of PCR technology. The biggest feature of PCR is that it can greatly increase a small amount of DNA, even if the synthesis amount of the target DNA molecule increases exponentially, so a small amount of genetic material can be ampli...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12P19/34
Inventor 曹雪雁李爱军史向阳
Owner DONGHUA UNIV
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