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Primer design method for amplifying low-content gene mutation DNA and application thereof

A technology for primer design and primer amplification, applied in biochemical equipment and methods, DNA preparation, recombinant DNA technology, etc., can solve the problems of limited detection ability, poor selectivity, and poor specificity, and achieve increased specificity , to solve the effect of insufficient detection sensitivity

Active Publication Date: 2009-12-30
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The primer defect of existing PCR technology design mainly contains: 1) specificity is not good, and the primer designed for the common method of gene mutation often produces non-specific amplification band
2) The selectivity is not good. In the background of a higher wild-type template, the detection ability for the lower content of gene mutation DNA is limited, and most of the tumor-causing mutations are somatic mutations, and the mutant cells are adulterated. mixed in wild-type cells, so the proposed DNA also contains a large amount of wild-type DNA

Method used

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  • Primer design method for amplifying low-content gene mutation DNA and application thereof
  • Primer design method for amplifying low-content gene mutation DNA and application thereof
  • Primer design method for amplifying low-content gene mutation DNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] In this example, a pair of primers were designed by the method of the present invention and combined with fluorescent quantitative PCR technology and double-circle probe technology to detect the gene mutation of K-ras gene 12 codon GGT>GAT.

[0039] Material:

[0040]

[0041]

[0042] 1. Preparation of samples and reference substances

[0043] 1). Sample processing and template extraction

[0044] Receive paraffin tissue samples from the clinic, cut the samples into 5-10 μm, add 1ml of xylene to dewax, collect the precipitate by centrifugation, add 1ml of absolute ethanol to the precipitate, dry at room temperature or 37°C, add proteinase K and Buffer ATL, 56 Digest and lyse at ℃ for 1 hour, incubate at 90°C for 1 hour, add 200ml Buffer AL and mix well, then add 200μl absolute ethanol and mix well, transfer the supernatant carefully to a QIA 2ml spin column, centrifuge at 6000×g (8000rpm) for 1min, add 500μl Buffer AW1, centrifuge at 6000×g (8000rpm) for 1min, ...

Embodiment 2

[0083] In this example, a pair of primers were designed by the method of the present invention in combination with fluorescent quantitative PCR technology and double-loop probe technology to detect the E746_A750del gene mutation in exon 19 of the EGFR gene.

[0084] Material:

[0085]

[0086] 1. Preparation of samples and reference substances

[0087] 1). Sample processing and template extraction

[0088] Receive paraffin tissue samples from the clinic, cut the samples into 5-10 μm, add 1ml of xylene to dewax, collect the precipitate by centrifugation, add 1ml of absolute ethanol to the precipitate, dry at room temperature or 37°C, add proteinase K and Buffer ATL, 56 Digest and lyse at ℃ for 1 hour, incubate at 90°C for 1 hour, add 200ml Buffer AL and mix well, then add 200μl absolute ethanol and mix well, transfer the supernatant carefully to a QIA 2ml spin column, centrifuge at 6000×g (8000rpm) for 1min, add 500μl Buffer AW1, centrifuge at 6000×g (8000rpm) for 1min, op...

Embodiment 3

[0128] In this example, a pair of primers were designed by the method of the present invention in combination with fluorescent quantitative PCR technology and double-circle probe technology to detect the T790M gene mutation in exon 21 of EGFR gene.

[0129] Material:

[0130]

[0131]

[0132] 1. Preparation of samples and reference substances

[0133] 1). Sample processing and template extraction

[0134]Receive paraffin tissue samples from the clinic, cut the samples into 5-10 μm, add 1ml of xylene to dewax, collect the precipitate by centrifugation, add 1ml of absolute ethanol to the precipitate, dry at room temperature or 37°C, add proteinase K and Buffer ATL, 56 Digest and lyse at ℃ for 1 hour, incubate at 90°C for 1 hour, add 200ml Buffer AL and mix well, then add 200μl absolute ethanol and mix well, transfer the supernatant carefully to a QIA 2ml spin column, centrifuge at 6000×g (8000rpm) for 1min, add 500μl Buffer AW1, centrifuge at 6000×g (8000rpm) for 1min, ...

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Abstract

The invention relates to a primer design method for amplifying low-content gene mutation DNA, and relates to a DNA amplification technology. The primers can be divided into four areas from 5' end to 3' end, namely a GC area, a 5' end matching area, a 3A area and a 3' end mutation matching area. The invention is characterized in that one segment of oligonucleotide tail unrelated to a target sequence to be amplified is added at the 5' tail end of one primer centered by each pair of primers, three A base segments are inserted into fifth to eighth oligonucleotides far from the tail end of 3', and mutation positions are arranged at first to fourth positions of the tail end of 3'. The primer design method can effectively and specifically amplify the target products when in higher wild-type DNA background under the condition of lower content mutation gene DNA, can carry out single weight or multiple weight PCR amplification and can be widely applied in the detection of nucleic acid.

Description

technical field [0001] The invention relates to a novel method for designing primers for nucleic acid amplification, in particular to a method for designing primers for amplifying low-content gene mutation DNA under the background of high wild-type DNA, and its application in nucleic acid detection. Background technique [0002] Polymerase Chain Reaction (Polymerase Chain Reaction, PCR) is a major leap in gene amplification technology, which was invented in 1985 by Professor Kary B Mullis of the Department of Genetics of PE Company in the United States. PCR technology was first applied to human β-globin DNA amplification and prenatal diagnosis of sickle cell anemia. Because this technology can specifically amplify a very small amount of target DNA by millions of times, it can greatly improve the analysis and detection capabilities of DNA molecules, and can detect single molecules or only 1 target DNA in 100,000 cells Molecular samples. Therefore, this technology has been w...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12P19/34C12Q1/68
Inventor 阮力何东华郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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