Chip and method for real-time PCR (polymerase chain reaction) gene detection at polygenic mutation site

A gene detection chip and site technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc.

Inactive Publication Date: 2012-04-25
杨楠 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0026] The object of the present invention is to provide a real-time PCR gene detection chip and its detection method for multiple gene mutation sites, which solves...

Method used

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  • Chip and method for real-time PCR (polymerase chain reaction) gene detection at polygenic mutation site
  • Chip and method for real-time PCR (polymerase chain reaction) gene detection at polygenic mutation site
  • Chip and method for real-time PCR (polymerase chain reaction) gene detection at polygenic mutation site

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Embodiment 1

[0073] Embodiment 1 Mutation detection of dystrophin gene (dystrophin)

[0074] 1. Synthesis and arrangement of primers

[0075] This embodiment adopts the gene mutation detection chip design of 96-well plate, and its primer probe is designed by primer 3 plus online software (http: / / www.bioinformatics.nl / cgi-bin / primer3plus / primer3plus.cgi), and Its specificity was verified in the UCSC database (http: / / genome.ucsc.edu / ).

[0076] The arrangement of primers on the 96-well plate is shown in Table 1:

[0077] Table 1 Primer layout design on 96-well plate

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] The primers in wells 1 to 2 were used for multiplex PCR to amplify 18 exons, wells 3 to 88 were used to detect mutations, and wells 89 to 96 were used as negative controls.

[0085] 2. Preparation of negative control DNA:

[0086] 1) Draw 2.5ml of normal human peripheral blood into a sodium citrate (1:9) anticoagulant tube.

[0087] 2) The blood...

Embodiment 2

[0111] Example 2 Detection of Rapid High-throughput Gene Mutations

[0112] Integrate the idea of ​​this chip design with the PCR system, and design 50 wells of 1cm in the future 2 The size of the chip, design a fluorescent real-time quantitative PCR system that can detect multiple chips at the same time, that is, develop a chip-PCR system that can detect multiple genes and multiple forms of mutation at the same time, which will greatly increase the throughput and reduce the size of the chip The cost of adding 1-5 μl of sample will be greatly reduced.

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Abstract

The invention discloses a chip and method for real-time PCR (polymerase chain reaction) gene detection at a polygenic mutation site. The gene detection chip comprises a porous PCR plate, wherein a PCR reaction system is arranged in each pore of the porous PCR plate; the PCR reaction system comprises a plurality of primer pairs for the specific amplification of target gene targeting sequences, fluorescent probes, PCR buffer solutions, dNTPs (deoxyribonucleotide triphosphates) and a GoldTaq enzyme; each primer pair contains a continuous nucleotide sequence composed of at least 15 continuous nucleotides in target gene exons; and fluorescent molecules of the fluorescent probes emit fluorescent signals in the PCR amplification process. The detection rate of the method on big genes can be up to 98% or above.

Description

technical field [0001] The invention belongs to the technical field of real-time detection of multi-gene mutation sites, and in particular relates to a real-time PCR gene detection chip for multi-gene mutation sites and a detection method thereof. Background technique [0002] A DNA mutation is any detectable heritable change in the genetic material, but does not include genetic recombination. Mutations can be passed on to daughter cells and even passed on to offspring, resulting in mutant cells or individuals. In a multicellular organism, if a mutation occurs only in somatic cells, then the mutation is not passed on to offspring. This type of mutation is called a somatic mutation. But if the mutation occurs in the germ cells of the human body, then the mutation can be passed on to the next generation through gametes, and the same mutation will be produced in the somatic cells and germ cells of the offspring individual. This mutation is called a germline mutation. Since t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 杨楠艾洪新杨林
Owner 杨楠
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