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Application of gold nanoparticle coated dendric macromolecular compound in polymerase chain reaction

A technology of dendritic macromolecules and gold nanoparticles, applied in the application field of polymerase chain reaction, can solve the problems of complex extraction and purification of SSB protein, short biological activity retention period, expensive commercial kits, etc. Preservation and accurate sample addition, good reagent uniformity, and improved specificity

Inactive Publication Date: 2011-02-16
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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  • Application of gold nanoparticle coated dendric macromolecular compound in polymerase chain reaction
  • Application of gold nanoparticle coated dendric macromolecular compound in polymerase chain reaction
  • Application of gold nanoparticle coated dendric macromolecular compound in polymerase chain reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Add Au / G5.NH 2 {(Au 0 ) 25 -G5.NH 2}DENPs are optimized for re-amplification PCR reactions with severe non-specific amplification.

[0046] 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.

[0047] In view of such PCR re-amplification that forms non-specific amplification, it is optimized by adding an effective amount of dendrimers to the PCR re-amplification system.

[0048] Specifically, the following steps are included:

[0049] 1. Prepare the PCR system for the first amplification.

[0050] The system composition is as follows:

[0051] Takara Ex Taq Enzyme (5U / μL)

0.125μL

10×PCR buffer (without Mg 2+ )

2.5μL

...

Embodiment 2

[0064] Add Au / G5.NH 2 {(Au 0 ) 50 -G5.NH 2}DENPs are optimized for re-amplification PCR reactions with severe non-specific amplification.

[0065] The reaction system and the specific process are the same as in Example 1, except that the added optimization agent is self-made {(Au 0 ) 50 -G5.NH 2}DENPs, with a concentration of 1.0 μg / μL and a molecular weight of 35860, needs to be diluted 1000 times in an ultra-clean bench before use.

[0066] Amplification results such as Figure 4 shown. From left to right is M: molecular weight marker (DL2000 of Takara company, respectively 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); 1 is not added {(Au 0 ) 50 -G5.NH 2}The re-amplification system of DENPs aqueous solution; 0 ) 50 -G5.NH 2}DENPs reamplification system; 7 is the blank control without template. It can be seen that adding {(Au 0 ) 50 -G5.NH 2}DENPs in the system of 0.4nM~0.49nM, the non-specific amplification in the PCR amplification results was significantly r...

Embodiment 3

[0068] Add Au / G5.NH 2 {(Au 0 ) 75 -G5.NH 2}DENPs are optimized for re-amplification PCR reactions with severe non-specific amplification.

[0069] The reaction system and the specific process are the same as in Example 1, except that the added optimization agent is self-made {(Au 0 ) 75 -G5.NH 2}DENPs, with a concentration of 1.0 μg / μL and a molecular weight of 40785, needs to be diluted 1000 times in an ultra-clean bench before use.

[0070] Amplification results such as Figure 5 shown. From left to right is M: molecular weight marker (DL2000 of Takara company, respectively 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); 1 is not added {(Au 0 ) 75 -G5.NH 2}The re-amplification system of DENPs aqueous solution; 0 ) 75 -G5.NH 2}DENPs reamplification system; 6 is the blank control without template. It can be seen that adding {(Au 0 ) 75 -G5.NH 2}DENPs 0.39nM ~ 0.51nM system PCR amplification results significantly reduced non-specific amplification, especially {(Au...

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Abstract

The invention relates to application of a gold nanoparticle coated dendric macromolecular compound in a polymerase chain reaction. The gold nanoparticle coated dendric macromolecular compound has the advantages of obvious amplification, low cost, wide application and easy storage of additives; the gold nanoparticle coated dendric macromolecular compound has great potential 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 application field of a dendrimer complex wrapping nano-gold particles, in particular to an application of a dendrimer compound wrapping nano-gold particles in polymerase chain reaction. Background technique [0002] Polymerase Chain Reaction (PCR) is a nucleic acid amplification technique that simulates natural DNA replication in vitro. The technology was invented by K.Mullis in 1985, and he himself won the Nobel Prize in Chemistry in 1995. PCR technology simulates the natural replication process of DNA in vivo, 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 suitable temperature extension. The thermal cycle is repeated: that is, at high temperature (95°C), the double-stranded target DNA to be amplified is denatured by heat to become two single-stranded DNA templat...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 曹雪雁陈晶晶史向阳
Owner DONGHUA UNIV