Polyase chain reaction optimization method based on arborized polymer

A chain reaction and optimization method technology, applied in the field of reaction, can solve the problems of complex extraction and purification of SSB protein technology, short biological activity retention period, expensive commercial kits, etc., to achieve easy storage, accurate sample addition, and uniformity of reagents Good, the effect of specificity improvement

Inactive Publication Date: 2009-01-14
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Polyase chain reaction optimization method based on arborized polymer
  • Polyase chain reaction optimization method based on arborized polymer
  • Polyase chain reaction optimization method based on arborized polymer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 adds polyamide-amine type dendrimer (G 4 -dendrimer) to optimize the re-amplification PCR reaction with severe non-specific amplification

[0044] 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.

[0045] 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.

[0046] Specifically, the following steps are included:

[0047] 1. Prepare the PCR system for the first amplification.

[0048] The system composition is as follows:

[0049] Takara Ex Taq Enzyme (5U / μL)

0.125μL

10×PCR buffer (without Mg 2+...

Embodiment 2

[0066] Embodiment 2 adds polyamide-amine type dendrimer (G 5 -dendrimer) to optimize the re-amplification PCR reaction with severe non-specific amplification

[0067] The reaction system and specific process are the same as in Example 1, except that the added optimization agent is self-made G 5 -Dendrimer, the concentration is 3.24μg / μL, it needs to be diluted 1000 times in the ultra-clean bench before use.

[0068] Amplification results such as figure 2 shown. From left to right: M: Molecular weight marker (DL2000 from Takara Company, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); 1 means no G 5 -Re-amplification system of dendrimer aqueous solution; 2, 3, 4, 5 are respectively added 0.65ng, 0.81ng, 0.88ng, 0.97ngG 5 -The re-amplification system of dendrimer; 6 is the blank control without adding template. It can be seen that adding G 5 -Dendrimer 0.65ng ~ 0.88ng system PCR amplification results significantly reduced non-specific amplification, especially G 5 -When the ...

Embodiment 3

[0069] Example 3 Adding 25% terminal aminoacetylated polyamido-amine type dendrimer (G 5 -dendrimer-25%Ac) to optimize the re-amplification PCR reaction with severe non-specific amplification

[0070] The reaction system and specific process are the same as in Example 1, except that the added optimization agent is self-made G 5 -dendrimer-25% Ac, the concentration is 2.84μg / μL, it needs to be diluted 1000 times in the ultra-clean bench before use.

[0071]Amplification results such as image 3 shown. From left to right: M: Molecular weight marker (DL2000 from Takara Company, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); 1 means no G 5 -Dendrimer-25% Ac aqueous solution re-amplification system; 2, 3, 4, 5 are respectively added 0.28ng, 0.99ng, 1.14ng, 1.28ng G 5 -dendrimer-25% Ac re-amplification system; 6 is the blank control without adding template. It can be seen that adding G 5 -Dendrimer-25% Ac 0.28ng ~ 1.14ng system PCR amplification results significantly reduced non...

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Abstract

The invention discloses a polymerase chain reaction optimizing method based on dendritic polymer, which belongs to the technical field of the biology. A dendritic polymer material is added into a polymerase chain reaction system as an additive agent to optimize the polymerase chain reaction, the dendritic polymer comprises multivalent nucleus combined with at least two dendritic branching covalences and a branch structure which can ensure the polymerization degree to achieve at least 2.0 through at least two generations of expansion. The dendritic polymer is diluted or concentrated and added into the polymerase chain reaction system in a gradient way, so as to achieve the range of the optimization purpose through actual amplification, and the range is the effective use amount range of the required dendritic polymer. The invention has the advantages that the optimization and amplification effects are remarkable, the cost of the additive agent is low, the amplified product is easy to be preserved, the application of the amplified product is wide, the specificity of the amplified product is obviously improved, and the output of the product in certain reactions is obviously enhanced.

Description

technical field [0001] The invention relates to a reaction method in the field of biotechnology, in particular to a polymerase chain reaction optimization method based on dendrimers. Background technique [0002] Polymerase chain reaction (PCR) is a nucleic acid amplification technique that mimics natural DNA replication in vitro. PCR technology is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers complementary to both ends of the target sequence. PCR is mainly composed of three steps of high-temperature denaturation, low-temperature annealing, and temperature-appropriate extension. The DNA generated in each cycle can become the template for the next cycle, and each cycle makes the difference between two artificially synthesized primers The copy number of the DNA-specific region is doubled, and the PCR product is 2 n The exponential form rapidly amplifies, and after 25 to 30 cycles, the gene can be amplified by 10 the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34
Inventor 曹雪雁杨文超张晓东李鑫辉胡钧
Owner SHANGHAI JIAO TONG UNIV
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