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Primer set and kit for screening human EGFR gene polymorphism and detecting method thereof

A gene polymorphism, primer set technology, applied in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., can solve problems such as staying and not being popularized

Active Publication Date: 2016-06-08
THE SECOND HOSPITAL OF DALIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the screening of genetic polymorphisms is still based on real-time quantitative PCR, and it is not popular.

Method used

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  • Primer set and kit for screening human EGFR gene polymorphism and detecting method thereof
  • Primer set and kit for screening human EGFR gene polymorphism and detecting method thereof
  • Primer set and kit for screening human EGFR gene polymorphism and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The specific process of screening EGFR19Del(2) polymorphism with 15 base deletions is as follows:

[0053] Before screening the polymorphism of the target gene segment, a pair of suitable primers (considering specificity and sensitivity) should be found according to the position of the missing base, that is, the BLAST tool in the NCBI website ( http: / / blast.ncbi.nlm.nih.gov / Blast.cgi ), to check the theoretical specificity of the primers. The cell line HCC827 containing the polymorphism and the cell line A549 not containing the polymorphism were used for amplification screening, and the specificity and sensitivity of the screening primers were confirmed again by agarose gel electrophoresis.

[0054]The amount and sequence of reagents and samples added during this exploration process: 12 μL double distilled water, 2 μL forward primer, 2 μL reverse primer (primer concentration is 10 μM), 2 μL sample DNA (concentration is 150 ng / μL) and 29.5 μL rebubble The rehydration b...

Embodiment 2

[0059] The specific process of the present invention in screening single nucleotide polymorphism EGFRL858R is:

[0060] Before screening the polymorphism of the target gene segment, a pair of suitable primers (considering specificity and sensitivity) should be found according to the position of the missing base, that is, the BLAST tool in the NCBI website ( http: / / blast.ncbi.nlm.nih.gov / Blast.cgi ), checked the theoretical specificity of the primers, and also utilized the website http: / / www6.appliedbiosystems.com / support / pnadesigner.cfm . Screened the PNA annealing temperature Tm value is 66 ℃). The cell line H-1975 containing the polymorphism and the cell line A549 not containing the polymorphism were used for amplification screening and the specificity and sensitivity of the primers were screened again by agarose gel electrophoresis.

[0061] The amount and sequence of adding reagents and samples in this exploration process: 10 μL double distilled water, 2 μL PNA, 2 μL sa...

Embodiment 3

[0067] The concrete implementation process of the sensitivity of screening the present invention (ARPS) is:

[0068] 1. The cell line DNA containing the polymorphism and the non-polymorphic cell line DNA are mixed, and the polymorphic cell line DNA (total DNA content is 300ng) is diluted to a concentration of 100%, 80%, 60%, respectively. 40%, 30%, 20%, 10%, and 0%. (100% or 300ng of DNA is all polymorphic cell line DNA, 80% or 300ng of DNA has 240ng of polymorphic cell line DNA, 60ng is non-polymorphic cell line DNA, and so on)

[0069] 2. According to the method described in Example 1, the following samples are detected respectively: except the positive control (+) in the kit and the negative control (-) without DNA, polymorphic cell line DNA (HCC-827 ) and normal target gene fragment cell line DNA (A-549 cell line), polymorphic cell line DNA (HCC-827) accounted for 100%, 80%, 60%, 40%, 30%, 20%, 10% % and 0%. Figure 3A shows that when the HCC-827 cell line DNA accounts f...

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Abstract

The invention belongs to the technical field of biology. Particularly, four molecular biology methods, namely, allele specific amplification, recombinase polymerase amplification, peptide nucleic acid combination DNA and SYBR GreenI coloration are combined. Lung cancer EGFR gene polymorphism is adopted as a screening object, and gene polymorphism screening methods are studied. A primer set and kit for screening human EGFR gene polymorphism and a detecting method thereof are obtained. The sequence of a primer is shown in SEQ ID NO.1-5. The primer set and kit can be used for screening of EGFR polymorphism and identifying patients suitable for targeted therapy. According to the method, no large equipment or a precision reagent of sidestream screening test paper is needed, great advantages are achieved on the aspects of specificity, sensitivity, simplificty, immediate screening and the like, and it is predicted that the method will be a fast screening method which is used for screening gene polymorphism, is consistent with the gene screening development wish and reliable, and has low cost benefits.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a rapid and rapid method combined with allele-specific amplification (ASA), recombinase polymerase amplification (RPA), peptide nucleic acid (PNA) combined with DNA technology and SYBRGreen I color development. New method for accurate screening of polymorphisms (single nucleotide polymorphisms and gene deletions) in the human EGFR gene. Background technique [0002] Fast, accurate and simple methods for screening genetic polymorphisms have been widely valued. The human genome is a very stable system. Different nations, groups and individuals have 46 chromosomes, the same number of genes and gene distribution, and basically the same nucleotide sequence. It is this stability of the genome structure that guarantees the commonality and stability of human beings as a species, and also determines that the current genome determination is meaningful, that is, representative. [0003] However...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6888C12Q2600/156C12Q2521/507C12Q2522/101C12Q2563/173
Inventor 王琪吕建新杜小慧刘元斌李恩成郭哲赵辉于若飞邝言斌
Owner THE SECOND HOSPITAL OF DALIAN MEDICAL UNIV