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Kit for detecting hot spot mutation sites in colorectal cancer PIK3CA gene

A mutation site, colorectal cancer technology, applied in the field of life science and biology, to improve the effect of chemotherapy, good specificity and high accuracy

Inactive Publication Date: 2012-12-12
杭州艾迪康医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this case, the Sanger method may not be the most suitable technique for DNA sequence analysis

Method used

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  • Kit for detecting hot spot mutation sites in colorectal cancer PIK3CA gene
  • Kit for detecting hot spot mutation sites in colorectal cancer PIK3CA gene
  • Kit for detecting hot spot mutation sites in colorectal cancer PIK3CA gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] (1) Sample extraction:

[0042]1.1 Draw 300 μl of blood, add 900 μl of red blood cell lysate, mix by inversion, and let stand at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 10,000rpm for 1 minute (if the maximum speed of the centrifuge is not allowed, centrifuge at 3,000rpm for 5 minutes), aspirate the supernatant, leave the white blood cell pellet, add 200μl buffer GA, shake until thoroughly mixed.

[0043] 1.2 Add 20 μl proteinase K solution and mix well.

[0044] 1.3 Add 200 μl buffer GB, mix thoroughly by inverting, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0045] 1.4 Add 200 μl of absolute ethanol, shake and mix well for 15 seconds, at this time flocculent precipitation may appear, centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0046] 1.5 Add the solution and flocculent prec...

Embodiment 2

[0060] Electrophoresis identification: 1.5% agarose gel electrophoresis, 140V, 20min, gel imaging system observation. The target fragments of exon 9 and exon 20 are 258bp and 250bp respectively, and the marker is DL2000.

Embodiment 3

[0062] Preparation of the single-stranded template: Add 3 μl of streptavidin-coated magnetic beads to 50 μl of each sample of the PCR product obtained from the aforementioned amplification and incubate at room temperature for 10 minutes. The PCR products combined with magnetic beads were incubated in 75% ethanol, 0.2mol / L NaOH solution and Tris eluent for 10 seconds respectively, and denatured to obtain single-stranded DNA after sufficient reaction. The single-strand released after binding to magnetic beads was suspended in 45 μl of annealing buffer (100 mM Tris-acetate pH7.75, 20 mM Mg-acetate) (containing 10 pmol of sequencing primers). Incubate at 80 °C for 2 min, then leave at room temperature for 5 min.

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Abstract

The invention discloses a kit detecting hot spot mutation sites in a colorectal cancer PIK3CA gene. The kit comprises an erythrocyte lysate, a whole blood DNA extraction reagent, an anhydrous ethanol, a PCR amplification reaction solution, a positive control, a negative control and a pyrosequencing reaction solution. The kit is characterized by comprising upstream and downstream primers 9F-Biotin and 9R for detecting exon 9 of the target gene and a sequencing primer 9RCX, and upstream and downstream primers 20F-Biotin and 20R for detecting exon 20 of the target gene and a sequencing primer 20RCX. The kit can be used to detect polymorphism of hot spot mutation sites (E542K, E545K and H1047R) in the colorectal cancer related PIK3CA gene, and has good specificity and high accuracy.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to a kit for detecting hotspot mutation sites (E542K, E545K and H1047R) of colorectal cancer PIK3CA gene, capable of detecting colorectal cancer PIK3CA gene mutation sites, The specificity is good, the accuracy is high, and the mutation detection rate can be improved. Background technique [0002] Colorectal cancer (CRC) is one of the most common malignant tumors. With the change of people's living environment and lifestyle, the incidence of CRC is increasing year by year. There are about one million new cases every year in the world, about Half a million patients died from it. The occurrence of human malignant tumors is related to many factors, has a complex molecular basis, and is a complex process of multi-step and multi-gene changes. The discovery of the oncogene PIK3CA provides a new direction for us to study the occurrence and development of tumors. F...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 薛群王淑一徐建成钟丹
Owner 杭州艾迪康医学检验中心有限公司
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