Detection probe of kRas gene mutation, liquid phase chip and detection method thereof

A probe and gene technology, applied in the field of kRas gene mutation detection probes, can solve problems such as hindering the detection of mutant genes, and achieve the effects of simple steps, convenient sampling, and avoiding uncertain factors

Active Publication Date: 2011-06-08
广州益善医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0021] In addition, due to the heterogeneity of the tumor, there may be only a very small amount of mutated genes in the free nucleic acid, while a large number of wild-type genes are contained. A large number of mixed wild-type genes will hinder the detection of mutated genes, so how to exclude wild-type sequences Interference with the detection of kRas gene mutation is a key problem to be solved first

Method used

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  • Detection probe of kRas gene mutation, liquid phase chip and detection method thereof
  • Detection probe of kRas gene mutation, liquid phase chip and detection method thereof
  • Detection probe of kRas gene mutation, liquid phase chip and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Preparation of liquid-phase chip for kRas gene mutation detection

[0078] 1. Probe sequence design and microsphere coating

[0079] Design specific oligonucleotide probes for the wild-type and mutant sequences of Codon12, 13 and 61 of kRas gene. The specific steps for coating each kind of microspheres are as follows:

[0080] (1) Take the mother liquid of microspheres (purchased from Luminex) vortex (vortex vibration) for 30s, and ultrasonic treatment for 1min;

[0081] (2) Take out 8ul microsphere mother liquor, containing 0.8×10 in total 5 —1.2×10 5 A microsphere into a 0.5ml centrifuge tube;

[0082] (3) Centrifuge at 15,000 rpm for 10 min, and discard the supernatant carefully;

[0083] (4) Add 10ul coupling solution (pH4.5), vortex for 30s, and ultrasonic treatment for 1min;

[0084] (5) Add 2ul of 2pmol / ul probe working solution;

[0085] (6) Add 2.5ul of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) working solution with a concentration of 5mg / ml, and inc...

Embodiment 2

[0096] Example 2. Codon 12 (Codon12) of kRas gene was introduced by PCR and wild-type excision site

[0097] Aiming at the sequence of codon 12 of the kRas gene, a pair of primers with BstOI restriction site was designed to amplify wild-type and mutant particles. The wild-type and mutant-type particles can be effectively removed by restriction enzymes, and the mutant-type will not be affected by the enzyme. Cut, so as to achieve the purpose of enriching mutants.

[0098] 1. PCR amplification

[0099] PCR reaction system:

[0100] Codon12 reaction system

[0101] PCR reaction conditions:

[0102]

[0103]

[0104] 2. BstOI digestion of PCR products:

[0105] The reaction system is as follows:

[0106] Sample system (Incubate at 60℃ for 2 hours)

[0107] Enzyme digestion result Figure 5 . The result of PAGE gel showed that the wild type of Codon12 was digested and digested completely, but the mutant type was not digested. This result proves the feasibility of PCR to introduce rest...

Embodiment 3

[0108] Example 3 Sensitivity experiment of kRas gene Codon12

[0109] In order to determine the sensitivity of Codon12, 1,3,9,27,81.243,729 copies of Codon12 mutant plasmid were used for detection, and each copy number was repeated 4 times.

[0110] 1. PCR amplification and restriction digestion

[0111] 1. The first round of PCR amplification

[0112] The PCR reaction system is the same as in Example 2.

[0113] PCR reaction conditions:

[0114]

[0115]

[0116] 2. BstOI digestion of PCR product:

[0117] The reaction system is the same as in Example 2.

[0118] 3. The second round of PCR amplification

[0119] PCR reaction system:

[0120] Codon12 reaction system

Per response (ul)

Sterilized ddH2O

28.8

5×Colorless GoTaq Flexi Buffer

10

2.5mM dNTP mix

2

MgCl 2 25mM

5

Codon12 primer F: K12EF (10uM)

1

Codon12 primer R: K12R-bio(10uM)

1

GoTaq Hot Start polymerase(5U / ul)

0.2

Template DNA

2

total capacity

50

[0121] PCR reaction conditions:

[0122]

[0123] Two, Luminex detectio...

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PUM

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Abstract

The invention discloses a nucleic acid probe, a liquid phase chip for detecting kRas gene mutation and a detection method thereof. The liquid phase chip for detecting the kRas gene mutation comprises a microsphere and a primer, wherein the microsphere is enveloped with a wild type and a mutation type probes which are modified with amidocyanogen and aim at kRas gene codons 12, 13 and / or 61; the primer is used for enlarging a corresponding mutant site which is enriched with the kPas gene codons 12, 13 and / or 61 and needs to be detected; and the tail end of the primer is provided with a biotin-labeled target sequence. The method is rapid, accurate and simple to operate, can synchronously detect a plurality of mutant sites in a hole and greatly improves detection efficiency. The liquid phase chip can be used for detecting the kPas gene mutation, can assist early diagnosis of pancreatic cancer, can accurately forecast effectiveness of molecular targeted drug treatment, is convenient for accurate medication in clinic and avoids the aging loss and the economic loss of unnecessary treatment.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a detection probe for kRas gene mutation, a liquid phase chip and a detection method thereof. Background technique [0002] 1. KRas gene mutation and early diagnosis of pancreatic cancer [0003] Ras gene is a common oncogene in human tumors. The Ras gene family is composed of kRas, hRas and nRas, and the homology among the members of the gene family can reach 85%. The protein encoded by the Ras gene is the P21 protein. The P21 protein is located on the inner surface of the cell membrane, has GTPase activity, and participates in the regulation system that transmits cell proliferation signals. The activated state is the GTP-bound state, and the inactive state is the GDP-bound state. The main part of its transformation into active oncogenes is the mutation of codons 12, 13, and 61. The activation of kRas gene has been studied in the occurrence of pancreatic cancer, and the poin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q2600/156C12Q1/6886
Inventor 吴诗扬任立芬许嘉森何嘉英杨惠夷林一群
Owner 广州益善医学检验所有限公司
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