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AS-PCR (allele-specific polymerase chain reaction) primer design method, gene mutation detection method and kit

A primer design and kit technology, applied in the field of molecular biology, can solve problems such as method-specific effects

Inactive Publication Date: 2015-05-13
江苏宏泰格尔生物医学工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] During the specific use process, the researchers found that the main limiting factor of the AS-PCR method in the prior art is that if the mutated site is a weakly mismatched base sequence, the specificity of the method will be affected

Method used

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  • AS-PCR (allele-specific polymerase chain reaction) primer design method, gene mutation detection method and kit
  • AS-PCR (allele-specific polymerase chain reaction) primer design method, gene mutation detection method and kit
  • AS-PCR (allele-specific polymerase chain reaction) primer design method, gene mutation detection method and kit

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Embodiment 1

[0066] The invention provides a kind of AS-PCR technique primer design method, comprises the following steps:

[0067] 1) Designing AS-PCR primers for the target sequence comprising the allelic mutation region to be tested;

[0068] 2) designing competitive blocking primers for the AS-PCR primers, and the competitive blocking primers are oligonucleotides that are reverse complementary to the AS-PCR primers.

[0069] Further, the length of the competitive blocking primer is consistent with that of the AS-PCR primer.

[0070] Further, bases that do not match the target sequence are introduced at the 2nd and / or 3rd bases at the 3' end of the AS-PCR primer.

[0071] The present invention also provides a kind of AS-PCR gene mutation detection method, comprises the following steps:

[0072] 1) Designing AS-PCR primers for the target sequence comprising the allelic mutation region to be tested;

[0073] 2) designing competitive blocking primers for the AS-PCR primers, which are ol...

Embodiment 2

[0078] In order to verify that the AS-PCR primer design method and gene mutation detection method of the present invention can be applied to the detection of allelic point mutations, the present invention provides an embodiment of the K-ras gene point mutation Gly12Asp (GGT>GAT) standard product fluorescence quantification PCR detection method.

[0079]According to the comparative analysis of wild gene sequence and mutant gene sequence of exon 12 of K-ras gene published by Cosmic data, AS-PCR primers are designed according to AS-PCR technology, and primers that can amplify a fragment of about 100bp are designed. The relevant parameters are: : Tm value 55.0°C-60.0°C, GC value 40.0%-60.0%, primer size 25±10bp. The 3' end of the upstream AS-PCR primer is located at the mutation site and is consistent with the mutant gene. In order to improve the specificity, a mismatched base is introduced at the third base of the 3' end. The designed The AS-PCR primer sequences are as follows: ...

Embodiment 3

[0101] In order to verify that the AS-PCR technology primer design method and gene mutation detection method of the present invention can be applied to the detection of allelic deletion mutations, the present invention provides an example of the fluorescence of EGFR gene exon 19 (E746-A750 deletion mutation) PCR detection method.

[0102] 1. Primer and Probe Design

[0103] According to the gene sequence of exon 19 (E746-A750 deletion mutation) of human epidermal growth factor receptor (EGFR) gene published by Cosmic data, use AS-PCR technology to design AS-PCR primers, and design a fragment that can amplify a fragment of about 100bp The relevant parameters of the primers are: Tm value 55.0°C-60.0°C, GC value 40.0%-60.0%, primer size 25±10bp. The 3' end of the upstream AS-PCR primer is located at the deletion mutation site and is consistent with the mutant gene. The designed AS-PCR primer sequence is as follows:

[0104] EGFR primer upstream sequence: AAATTCCCGTCGCTATCAAAAC ...

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Abstract

The invention relates to the field of molecular biology, in particular to an AS-PCR (allele-specific polymerase chain reaction) primer design method, a gene mutation detection method and a kit. The AS-PCR primer design method comprises the following steps: (1), an AS-PCR primer is designed for a target sequence containing a to-be-detected allele mutation region; (2), a competition blocking primer is designed for the AS-PCR primer and adopts oligonucleotide in reverse complement with the AS-PCR primer. According to the gene mutation detection method, the AS-PCR primer and the competition blocking primer have a PCR amplification reaction. The kit comprises the AS-PCR primer, the competition blocking primer, a TaqMan probe, an internal control primer, an internal control probe, polymerase, dNTP (deoxynucleotide) and a buffer solution. The AS-PCR primer design method, the gene mutation detection method and the kit have the advantage that the difference of specificity of the AS-PCR primers on mutation sites due to strong and weak mismatch of the mutation sites is reduced effectively.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an AS-PCR technique primer design method, a gene mutation detection method and a kit. Background technique [0002] Gene mutation is a change in gene structure caused by the addition, deletion or substitution of base pairs in DNA molecules. A wild-type gene becomes a mutant gene by mutation. From the perspective of molecular biology, malignant tumors can be regarded as genetic diseases, which are the result of gene mutations caused by DNA damage on certain chromosomes, resulting in uncontrolled cell growth, lack of differentiation and abnormal proliferation, and can invade normal tissues and organs , and eventually spread throughout the body. A tumor is a single cell that grows abnormally due to a genetic mutation, and is formed from the cloned descendants of this abnormally growing cell. During tumor progression, additional genetic mutations often occur in the tumor cell popu...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 张翼飞汤世博
Owner 江苏宏泰格尔生物医学工程有限公司
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