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Multiplex polymerase chain reaction method and its application

A technology of chain reaction and polymerase, applied in the field of multiple polymerase chain reaction

Active Publication Date: 2020-06-05
SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the use of graphene oxide in multiplex PCR technology

Method used

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  • Multiplex polymerase chain reaction method and its application
  • Multiplex polymerase chain reaction method and its application
  • Multiplex polymerase chain reaction method and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1: Effects of different concentrations of graphene oxide on the multiple amplification of human genome cancer genes

[0116] 1. Purpose of the experiment

[0117] Using the human genome as a template, 8 cancer-related genes (Shuichi Hoshika, Fei Chen, Nicole A. Leal, et al. Artificial Genetic Systems: Self-Avoiding DNA in PCR and Multiplexd PCR[J].Angew.Chem.Int . Ed. 2010, 49, 5554–5557) for multiplex amplification in one PCR reaction. Comparison of the effect of adding different concentrations of graphene oxide on multiplex PCR.

[0118] 2. Experimental materials and methods

[0119] Name of cancer gene to be amplified, primer sequence and product length

[0120]

[0121]

[0122] Composition of PCR solution system (25μL):

[0123]

[0124] PCR steps and conditions: (1) Pre-denaturation at 95°C for 5 minutes; (2) Pre-denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute, extension at 72°C for 1 minute and 30 seconds; repeat 40 cycles; ...

Embodiment 2

[0129] Embodiment 2: graphene oxide is to the improvement of multiplex PCR sensitivity

[0130] 1. Purpose of the experiment

[0131] Using the human genome as a template, 8 cancer-related genes were multiplexed in one PCR reaction. Different concentrations of templates were used to amplify, and the sensitivity of multiplex PCR without adding GO and adding an appropriate amount (250ng) of graphene oxide was compared.

[0132] 2. Experimental materials and methods

[0133] The name of the cancer gene to be amplified, the primer sequence and the length of the product are the same as in Example 1.

[0134] Composition of PCR solution system (25μL):

[0135]

[0136] PCR steps and conditions: with embodiment 1;

[0137] Electrophoresis analysis conditions: with embodiment 1.

[0138] 3. Experimental results

[0139] The amplified products after multiplex PCR were detected by agarose gel electrophoresis, and the results were as follows: figure 2 shown. M represents DNA ...

Embodiment 3

[0140] Example 3: Graphene oxide inhibits non-specific amplification in multiplex PCR and the specificity of multiplex PCR at different annealing temperatures

[0141] 1 Experiment purpose

[0142] Using Lambda DNA as a template, 8 primer pairs were used to simultaneously amplify 8 different regions on Lambda DNA. The enhancement effect of graphene oxide on multiplex PCR specificity was compared at various annealing temperatures.

[0143] 2 Experimental materials and methods

[0144] Primer sequences used for amplification, product length and amplified region

[0145]

[0146]

[0147] Composition of PCR solution system (25μL):

[0148]

[0149] PCR steps and conditions: (1) Pre-denaturation at 95°C for 5 minutes; (2) Pre-denaturation at 95°C for 30 seconds, annealing at different temperatures (52, 60, 68, 72°C) for 1 minute and 30 seconds, and extension at 72°C for 1 minute Minutes and 30 seconds; repeat 40 cycles; (3) 68°C extension for 10 minutes.

[0150] 3. ...

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Abstract

The invention provides a multiple polymerase chain reaction method and an application thereof. The method comprises the following steps: A) providing a polymerase chain reaction system, wherein the reaction system contains a template to be amplified, a primer pair used for amplification, polymerase, oxidized graphene and a reagent required for performing the polymerase chain reaction; B) performing the polymerase chain reaction to obtain a reaction mixture containing an amplified product, wherein the amplified product comprises amplification products respectively corresponding to the primer pair; and C) performing electrophoresis and / or sequencing detection on the reaction mixture in the step B). According to the method, all specific amplified products can be accurately amplified with high efficiency in a multiple PCR reaction system containing low copy-amount complex template and are capable of easily generating non-specific amplification. The multiple polymerase chain reaction method can be widely used in different detection fields.

Description

technical field [0001] The present invention relates to a multiplex polymerase chain reaction method, in particular to the application of the multiplex polymerase chain reaction method in efficiently and accurately amplifying target nucleic acid sequences in low copy number and highly complex templates. Background technique [0002] Polymerase chain reaction (PCR) can use a small amount of DNA as a template, and enrich the target DNA fragments in a short time (several hours) in a manner of exponential amplification. Multiplex PCR can simultaneously amplify multiple genes in one reaction. Compared with conventional PCR technology, it has the advantages of high efficiency, time saving, and sample saving. Therefore, this technology has become a quick and easy gene detection in clinical and basic research. method. However, in the multiplex PCR reaction system, high concentrations of multiple primers interact with each other, which seriously affects the specificity and uniformit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686
Inventor 张琛张东华张益胡钧
Owner SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI