PNA, probe, primer and method for detecting K-ras gene mutation

A probe and gene technology, applied in the field of PNA to detect K-ras gene mutation, can solve the problems of cumbersome steps and achieve the effect of simple operation, high sensitivity and high specificity

Inactive Publication Date: 2009-05-06
SHANGHAI CHANGHAI HOSPITAL
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  • Summary
  • 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 i

Method used

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  • PNA, probe, primer and method for detecting K-ras gene mutation
  • PNA, probe, primer and method for detecting K-ras gene mutation
  • PNA, probe, primer and method for detecting K-ras gene mutation

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1 Carrying out real-time quantitative PCR detection on the plasmid standard substance of known mutation amount

[0050] 1. Main source of reagents:

[0051] 1.1 Designing PNAs

[0052] PNA is a PNA designed for the 12th and 13th codons of the wild-type K-ras gene, comprising at least 12 consecutive bases of SEQ ID NO: 1 or its complementary sequence, and the preferred sequence of the PNA is Ac-ACGCCACCAGCTC-Lysine-COOH (SEQ ID NO: 1) ID NO: 1), synthesized by South Korea panagene company.

[0053] 1.2 Design of Tagman MGB probe

[0054] The Tagman MGB probe hybridizes to the wild-type K-ras gene target sequence, the probe sequence comprising at least 15 consecutive nucleotides of SEQ ID NO: 2 or its complement. The probe is preferably an oligonucleotide sequence shown in SEQ ID NO: 2, i.e. FAM-AGCTCCAACTACCACAAG-MGB (SEQ ID NO: 2), and its two ends are respectively labeled with a reporter group FAM and a quencher group MGB. Needles were synthesized by Shangh...

Embodiment 2

[0105] Example 2 Detection of K-ras gene mutation in clinical samples (taking blood and stool samples as examples)

[0106] 1. Collect the feces and blood of patients with clinical pancreatic cancer, chronic pancreatitis and normal people, and extract DNA.

[0107] 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.

[0108] 3. Fecal DNA extraction method: Qia...

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Abstract

The invention discloses PNA, probe and primer for testing K-ras genetic mutation, and a method for quantitatively testing the K-ras genetic mutation in real time by the PNA, the probe and the primer. The PNA is hybridized with the 12th codon and/or the 13th codon in the first exon of wild K-ras gene; and the PNA is combined with real-time quantitative PCR to quantitatively test the mutation amount of the K-ras gene in real time. The method has the advantages of simple and convenient operation, high sensitivity and high specificity, and can be applied to early auxiliary diagnosis for pancreatic cancer and other diseases.

Description

technical field [0001] The invention relates to the detection of K-ras gene mutation, in particular to a PNA, probe, primer and method for detecting 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 pa...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 李兆申林寒高军龚燕芳杜奕奇
Owner SHANGHAI CHANGHAI HOSPITAL
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