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Application of graphene in polymerase chain reaction as reinforcing agent

A technology of chain reaction and polymerase, applied in the field of graphene materials, to achieve the effect of simplifying steps, ensuring accuracy, and satisfying a large number of amplification

Inactive Publication Date: 2013-03-20
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high-efficiency PCR amplification remains a challenge as different methods have different constraints

Method used

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  • Application of graphene in polymerase chain reaction as reinforcing agent
  • Application of graphene in polymerase chain reaction as reinforcing agent
  • Application of graphene in polymerase chain reaction as reinforcing agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Mix 2g of natural graphite powder and 1g of sodium nitrate with 46mL of concentrated sulfuric acid in an ice bath, then add 6g of potassium permanganate, stir the mixture in an ice bath for 2 hours, then transfer to an oil bath at 35±5°C React for 30 minutes, the solution is brown and viscous, then slowly add 92mL of pure water to the mixture, raise the temperature to 95±5°C and continue the reaction for 3 hours, the mixture turns from brown to bright yellow, and finally add 6mL H with a mass fraction of 30% 2 O 2The solution is used to neutralize the unreacted potassium permanganate. After the above solution is cooled to room temperature, it is filtered with suction and the filter cake is repeatedly washed with pure water, dried in vacuum at 60° C. overnight to obtain graphite oxide. A certain mass of graphite oxide was weighed and dispersed into pure water, ultrasonicated for 5 hours to dissolve it completely, and then centrifuged at a low speed of 4000rpm...

Embodiment 2

[0033] Example 2: The plasmid DNA template pET-32a was purchased from Novegan Company, and the length of the amplified target product was 300bp. The primer sequences used for amplification were: forward primer P15'-GCC ATG GAT CAC AAG CAT CAT GAT CAC-3', reverse primer P25'-GCC TCG AGG CAG AAA CAG TCG CAG T. The PCR system is 1×PCR buffer (20mM Tris-HCl, pH8.8, 10mM KCl, 16mM (NH 4 ) 2 SO 4 ,2mM MgSO 4 ,1~0.02mg mL -1 BSA, 0.1%TritonX-100), 0.2mM dNTPs, 0.4μM each of forward and reverse primers, 20ngμL -1 Plasmid DNA, 0.1U μL -1 PfuDNA polymerase, and finally the system volume was supplemented to 25 μl by graphene oxide or reduced graphene and sterilized double distilled water. In multiple rounds of PCR, the template DNA in each cycle was replaced by 0.4 μl of PCR product from the previous round. PCR conditions are: 1) pre-denaturation at 94°C for 5 min, 2) denaturation at 94°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 1 min, this step was repeated 30 ...

Embodiment 3

[0042] Example 3: The human genome template was collected from the blood of volunteers and used to amplify a 794bp DNA fragment located in the 12sRNA and 16sRNA regions of mitochondria. The amplification primers were forward primer 5'-AAC TAC GAT AGC CCT TATGAA-3', and reverse primer 5'-GGC CTACTATGG GTG TTAAA-3'. The PCR system is 1×PCRbuffer (20mM Tris-HCl, pH8.8, 10mM KCl, 16mM (NH 4 ) 2 SO 4 ,2mM MgSO 4 ,1~0.02mg mL -1 BSA, 0.1%TritonX-100), 0.2mM dNTPs, 0.4μM each of forward and reverse primers, 20ngμL -1 Genomic DNA, 0.1UμL -1 Pfu DNA polymerase, finally supplemented with graphene oxide or reduced graphene and sterilized double distilled water to make up the system volume to 25 μL. PCR conditions were 1) pre-denaturation at 94°C for 5 min, 2) denaturation at 94°C for 45 s, annealing at 52°C for 45 s, extension at 72°C for 90 s, this step was repeated 34 times, and 3) extension at 72°C for 10 min. Then use 1.5% EB stained agarose gel electrophoresis to detect the P...

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Abstract

An application of graphene in polymerase chain reaction as a reinforcing agent relates to a graphene material. An improved Hummers method is utilized to synthesize graphite oxide, and hydrazine hydrate is utilized to synthesize graphene. A plasmid is used as a template to carry out polymerase chain reaction and multi-round polymerase chain reaction, the graphene can increase the specificity of the polymerase chain reaction within 14 mu gmL-1, and a target product with strong specificity can even be obtained through 8-round polymerase chain reaction. A clinical blood sample deoxyribonucleic acid (DNA) is taken as a template to carry out the polymerase chain reaction, and a polymerase chain reaction (PCR) reaction system which is added with the graphene can obtain a product with single specificity and can still obtain the target product with the strong specificity within a scope between 25 DEG C and primer annealing temperature. The amplification products which are added with the graphene do not influence the primer sequence and length through sequencing tests, and the graphene can be used as a polymerase chain reaction reinforcing agent with good performance for polymerase chain reaction.

Description

technical field [0001] The invention relates to graphene materials, in particular to the application of graphene as a reinforcing agent in polymerase chain reaction. Background technique [0002] Since its invention by Kary Mullis in 1985, polymerase chain reaction (PCR) has become an important technical method for DNA amplification in vitro in modern life sciences. PCR can generate a large number of target DNA fragments through exponential amplification within a few hours with a small amount of DNA template. However, non-specific DNA fragments are often generated during this process, which reduces the efficiency of PCR. It has been reported before that different additives are used to improve the efficiency of PCR. However, high-efficiency PCR amplification remains a challenge because different methods have different restrictive conditions. Although controversial, it has been reported that materials with large specific surface area and excellent thermal and electrical con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 孙莉萍胡楠贾静翁建
Owner XIAMEN UNIV
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