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Quantitative determination method for K-ras gene mutation

A quantitative detection and gene technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of cumbersome steps and achieve the effect of simple operation, high specificity and high sensitivity

Inactive Publication Date: 2009-05-20
SHANGHAI CHANGHAI HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] (1) The steps are cumbersome and it takes 2 days to complete the identification;
[0006] (2) It can only be used for qualitative research, that is, it can only be determined whether there is a K-ras gene mutation. If quantitative research is required, other methods must be used for further testing;
[0007] (3) False positives caused by incomplete first round of enzyme digestion
[0014] (1) Each specific primer or probe can only be identified for a certain type of gene mutation, and there are more than ten types of mutations in the 12th and 13th codons of the K-ras gene, which need to be detected one by one;
[0015] (2) There may be false positives due to single base mismatches

Method used

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  • Quantitative determination method for K-ras gene mutation
  • Quantitative determination method for K-ras gene mutation
  • Quantitative determination method for K-ras gene mutation

Examples

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Embodiment 1

[0039] Example 1 Carrying out real-time quantitative PCR detection on the plasmid standard substance of known mutation amount

[0040] 1. Main source of reagents:

[0041] 1.1 Designing PNAs

[0042] The PNA is designed for the 12th and 13th codons of the wild-type K-ras gene, and its sequence is Ac-ACGCCACCAGCTC-Lysine-COOH (SEQ ID NO: 1), which was synthesized by Panagene Company in South Korea.

[0043] 1.2 PCR primers

[0044] The present invention is used for the primer of amplification K-ras gene target sequence, comprises following primer pair:

[0045] Upstream primer F1: 5'-TACTGGTGGAGTATTTGATA-3' (SEQ ID NO: 2);

[0046] Downstream primer R1: 5'-CAAGATTTACCTCTATTGTT-3' (SEQ ID NO: 3);

[0047] The above PCR primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.

[0048] 1.3 TA cloning kit

[0049] TA cloning kit (Target Clone) was purchased from TOYOBO Company.

[0050] 1.4 Plasmid extraction kit

[0051] The plasmid extraction kit was...

Embodiment 2

[0095] Example 2 Detection of K-ras gene mutation in clinical samples (taking blood and stool samples as examples)

[0096] 1. Collect the feces and blood of patients with clinical pancreatic cancer, chronic pancreatitis and normal people, and extract DNA.

[0097] 2. Blood DNA extraction method: ① anticoagulated whole blood 0.5 ~ 12ml; ② add 500 ~ 700ulTT buffer, shake vigorously, centrifuge at 12000 rpm for 5 minutes in a low temperature centrifuge; ③ collect the precipitate, repeat steps 2 and 3 , until the precipitate is colorless; ④Add 200ul PCR lysate A; ⑤37 ℃ water bath overnight; Add 200ul of chloroform to the supernatant, mix thoroughly, and centrifuge at 12000 rpm for 10 minutes; / centrifuge for 10 minutes; ⑨discard the supernatant and add 1ml of 70% ethanol to wash, 12000 rpm / centrifuge for 5 minutes; ⑩discard the supernatant, air-dry naturally, dissolve the precipitate with 100ulTE solution, aliquot and store at -80°C.

[0098] 3. Fecal DNA extraction method: Qia...

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Abstract

The invention discloses a quantitative detection method of K-ras gene mutation, comprising the following steps: a PNA which is hybridized with a twelfth and / or thirteenth codon in a first exon of a wild-typed K-ras gene is designed; SYBR-RT-PCR quantitative detection is carried out to the mutant-typed and wild-typed standard products with known copy numbers respectively with the PNA for obtainingstandard and melting curves; SYBR-RT-PCR quantitative detection is carried out to the sample nucleic acid to be detected with the PNA; and after the sample nucleic acid to be detected is judged to bewild type or mutant type or mutant / wild hybrid type according to the melting curve, the K-ras gene mutation quantity of the sample nucleic acid to be detected is obtained according to the standard curve. The method of the invention has simple operation, high sensitivity and high specificity, and can be applied to early auxiliary diagnosis on diseases such as pancreatic cancer, etc.

Description

technical field [0001] The invention relates to the detection of K-ras gene mutation, in particular to a quantitative detection method of K-ras gene mutation. Background technique [0002] K-ras gene (GenBank: NC_000012) mutation plays an important role in the occurrence and development of pancreatic cancer. It has been reported that point mutations in codon 12, 13 or 61 of the K-ras gene can be detected in pancreatic cancer tissue, pancreatic juice or feces, and blood (Takahashi, et al. Gastrointestinal Endo 2005.61: 76-9; Luigi L, et al. Oncogene 2002.21:4301-6; TadaM, et al. Cancer Res 1993.53:2472-4; Lu XH, et al.Natl Med J China2001.81:1050-3; N van Heek, et al.J.Clin.Pathol. 2005.58: 1315-1320). [0003] Studies have shown that detection of K-ras gene mutation in somatic DNA can be used as a method for early diagnosis of pancreatic cancer. However, in some experiments, it was found that K-ras gene mutations also exist in benign diseases such as chronic pancreatitis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 李兆申高军林寒杜奕奇龚燕芳
Owner SHANGHAI CHANGHAI HOSPITAL
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