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Method and kit for identifying free DNAs in serum by adopting mass spectrometer and applications of method and kit

A technology of mass spectrometer and kit, applied in Kras gene to detect free DNA in serum, mass spectrometer to identify free DNA in serum, it can solve the problems of unsatisfactory detection speed, false positive and so on, so as to reduce the power consumption of the instrument and reduce the Overall size, effect of improving sensitivity

Inactive Publication Date: 2014-02-26
BIOYONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the fluorescent quantitative PCR method involves fluorescent dyes, which are prone to false positives.
[0006] The Chinese invention application document with application number 200910177851.2 provides a method for detecting nucleotide mutation sites of KRAS gene and / or BRAF gene, which improves the sensitivity and accuracy of detection to a certain extent, but its detection speed Still can not meet the needs of the status quo

Method used

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  • Method and kit for identifying free DNAs in serum by adopting mass spectrometer and applications of method and kit
  • Method and kit for identifying free DNAs in serum by adopting mass spectrometer and applications of method and kit
  • Method and kit for identifying free DNAs in serum by adopting mass spectrometer and applications of method and kit

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Embodiment 1

[0095] A method for mass spectrometer identification of free DNA in serum, comprising the steps of:

[0096] (1) PCR reaction: the Kras gene in the free DNA was amplified by PCR to obtain a PCR product containing the 12-codon and 13-codon regions of the Kras gene; The PCR amplification is carried out with PCR primer 1 and / or PCR primer 2;

[0097] (2) Purification of PCR products: Purify the PCR products obtained in step (1) to reduce interference to subsequent reactions;

[0098] (3) Single-base extension: use iPLEX Pro enzymes, ddATPs, ddCTPs, ddTTPs and specific extension primers to perform multiple single-base extensions on the purified PCR product obtained in step (2), and the extension primers are at the corresponding SNP positions A nucleotide is extended at the point, and the nucleotide is complementary to the genotype at the SNP site; the specific extension primers are SEQ ID No: 3, SEQ ID No: 4, and SEQ ID No: 5;

[0099] (4) Purification of extension products: Pur...

Embodiment 2

[0170] A detection kit, which is used in the method for mass spectrometer identification of free DNA in serum, said kit comprising:

[0171] (1) PCR reaction reagents, including: PCR primers, high temperature resistant DNA polymerase, dNTPs, PCR reaction buffer, buffer; the PCR primers are SEQ ID No: 1 and SEQ ID No: 2;

[0172] (2) PCR product purification reagents;

[0173] (3) Single-base extension reaction reagents, including: extension primers, high-temperature-resistant single-base extension enzymes, ddNTPs, and extension reaction buffers; the high-temperature-resistant single-base extension enzymes are iPLEX Pro enzymes; the extension primers It is any one or more of SEQ ID No: 3, SEQ ID No: 4, and SEQ ID No: 5.

[0174] In this embodiment, the high temperature-resistant DNA polymerase is Taq enzyme; the PCR reaction buffer is Tris-CL buffer; the 5' end of the PCR primer contains a protective base, and the protective base sequence for ACGTTGGATG.

[0175] In this emb...

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Abstract

The invention belongs to the field of biotechnology, and relates to a method and a kit for identifying free DNAs in serum by adopting a mass spectrometer and applications of the method and the kit. The method comprises the steps of PCR reaction, PCR product purification, single base extension, extension product purification and detection by adopting the mass spectrometer; the kit comprises a PCR reaction reagent, a purifying reagent and a single base extension reaction reagent. Compared with the prior art, the method and the kit provided by the invention have the advantages of high sensitivity, strong specificity, simplicity and safety, high speed, high flux, and the like, and a relatively large amount of time is saved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for identifying free DNA in serum by a mass spectrometer, a kit and an application thereof, specifically using PCR technology, single-base extension technology and mass spectrometry technology, and selecting Kras gene to detect free DNA in serum. DNA testing methods. Background technique [0002] Free circulating nucleic acid is an extracellular free state nucleic acid that exists in human body fluids. The molecular size of free circulating DNA is between 500bp and 30kb. Free DNA in peripheral blood of normal people mainly comes from nuclear DNA and mitochondrial DNA, and its content is relatively small, while free DNA in peripheral blood of cancer patients mainly comes from tumor cells. [0003] In recent years, with the deepening of research on cell-free nucleic acid, the detection of cell-free DNA has made many progresses in disease monitoring, fetal prenatal diagnosis an...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6872C12Q2565/627
Inventor 张学记马庆伟张海燕林燕
Owner BIOYONG TECH
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