Method for using polyethyleneimine to strengthen sensitivity and specificity of PCR gene splicing by overlap extension

A polyethylenimine and overlapping extension technology, applied in the field of genetic engineering, can solve problems such as no standardized reaction conditions

Inactive Publication Date: 2017-02-01
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, as a technical system without standardized reaction conditions, there are still many problems to be solved in the application process

Method used

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  • Method for using polyethyleneimine to strengthen sensitivity and specificity of PCR gene splicing by overlap extension
  • Method for using polyethyleneimine to strengthen sensitivity and specificity of PCR gene splicing by overlap extension
  • Method for using polyethyleneimine to strengthen sensitivity and specificity of PCR gene splicing by overlap extension

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] In the present invention, the oligonucleotide primer design process is as follows:

[0037] 1. Software used: GenSearch

[0038] 2. The results output by GenSearch are shown in the table below:

[0039] The designed synthetic primers of HU-βgene are shown in Table 1:

[0040]

[0041]

[0042] Table 1

Embodiment 2

[0043] Embodiment 2: PEI preparation of different concentrations

[0044] ① Draw 100ul from the PEI stock solution provided by FluKa and add 400ul of distilled water to obtain a volume percentage concentration of 10 -1 The PEI solution; PEI stoste is the aqueous solution that volume percent concentration is 50%;

[0045] ②From volume percentage concentration to 10 -1 Draw 100ul in the PEI solution and add 900ul of distilled water to obtain a volume percentage concentration of 10 -2 PEI solution;

[0046] ③From volume percentage concentration to 10 -2 Draw 100ul in the PEI solution and add 900ul of distilled water to obtain a volume percentage concentration of 10 -3 PEI solution;

[0047] ④ Carry out the same operation in sequence to obtain a volume percentage concentration of 10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 、10 -11 、10 -12 、10 -13 PEI solution.

Embodiment 3

[0048] Example 3: Effects of Different Concentrations of PEI on the Synthesis of Overlap Extension PCR

[0049] The designed HU-β primers were dissolved in 10mM Tris-HCl (pH 8.5) to a concentration of 100μM, then all the primers were mixed and diluted with distilled water to obtain a oligonucleotide chain mixture with a concentration of 1μM, and then the mixture was washed with distilled water Dilute 10 times. Used as a template in the PCR reaction system, and to a final concentration of all primers of 25 nM.

[0050] Prepare 20 μl of PCR reaction solution according to Table 2.

[0051]

[0052]

[0053] Table 2

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Abstract

The invention discloses a method for using polyethyleneimine to strengthen sensitivity and specificity of PCR gene splicing by overlap extension. The method comprises the steps of designing an HU-Beta primer sequence which needs to be synthesized, and obtaining a blended overlapping oligonucleotides template, wherein every sequence contains 25bp overlapping oligonucleotides template so as to cover the entire DNA(200-1500 basic group) sequence; adding DNA polymerase, head and tail primer, and the solution of blended overlapping oligonucleotides which is used as a template in the last step into into PEI ,the volume of which should be 10<-5>-10<-13> that of the DNA polymerase, head and tail primer, and the solution of blended overlapping oligonucleotides; carrying out repeated circulation according to three steps of melting, annealing, extending of conventional PCR, synthesizing and amplifying the target nucleic acid segment. The method for using polyethyleneimine to strengthen sensitivity and specificity of gene splicing by overlap extension can specifically and efficiently synthesize and amplify the target nucleic acid sequences.

Description

[0001] 【Technical field】 [0002] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for improving the sensitivity and specificity of PCR gene synthesis by utilizing a water-soluble cationic polymer polyethyleneimine (PEI). [0003] 【Background technique】 [0004] The artificial synthesis of DNA has always been an important part of gene research. Overlap extension PCR (gene splicingby overlap extension: SOE-PCR) technology is a simple and rapid gene synthesis method. [0005] Overlap extension PCR technology is based on the nucleotide sequence of the gene, divides the target gene into multiple primers ranging from 50-90bp, and synthesizes them in segments. As a template, each fragment is combined into a target fragment through several rounds of continuous PCR reactions. [0006] The system of overlap extension PCR technology has become mature day by day and has become a common method in molecular biology research. Howev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1031C12Q2527/125
Inventor 张云威戚智青肖桂清肖志勇刁勇
Owner HUAQIAO UNIVERSITY
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