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Breast cancer PIK3CA mutant gene and application thereof

A technology for mutating genes and breast cancer, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem that PIK3CA gene breast cancer has not been reported, and achieve good practical application value

Active Publication Date: 2016-01-13
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The S553fs*7 mutation is one of the PIK3CA gene mutations, but the PIK3CA gene S553fs*7 mutation has not been reported in breast cancer

Method used

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  • Breast cancer PIK3CA mutant gene and application thereof
  • Breast cancer PIK3CA mutant gene and application thereof
  • Breast cancer PIK3CA mutant gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: DNA Extraction in Breast Cancer and Paracancerous Tissues

[0022] 1. Experimental materials

[0023] TES buffer (wash a 50mL centrifuge tube, after high temperature and high pressure sterilization, add 0.5mL of 1M Tris-HCL, 1mL of 0.5M EDTA and 2.5mL of 10% SDS, add ultrapure water to make up to 50mL, adjust the pH to 8.0 Store at room temperature), 20mg / mL proteinase K, 3M sodium acetate (pH=5.2), TEBuffer, phenol, chloroform, isoamyl alcohol, absolute ethanol, TE solution.

[0024] 2. Experimental method

[0025] 2.1 Thaw the tissue block, cut about 0.5g of tissue, put it into a 1.5mL centrifuge tube, and cut it into pieces;

[0026] 2.2 Add 0.45mL LTES buffer and mix well, then add 50μL SDS (10%), 5.0μL proteinase K (20mg / mL), mix well, digest at 56°C for 4-6h, shake once every 2h until the solution is clear;

[0027] 2.3 Place it at room temperature, add an equal volume of saturated phenol (500 μL), invert to mix, 12000 rpm, centrifuge for 10 minute...

Embodiment 2

[0033] Embodiment 2: DNA extraction in blood sample

[0034] 1. Experimental materials

[0035] White blood cell lysate, red blood cell lysate, proteinase K (10mg / mL), phenol, chloroform, isoamyl alcohol, absolute ethanol, TE solution.

[0036] 2. Experimental method

[0037]2.1 Transfer 4mL of fresh blood to a 15mL centrifuge tube, add red blood cell lysate, mix upside down, centrifuge at 4000g at 4°C for 15min;

[0038] 2.2 Remove the supernatant and wash the blood repeatedly 2-3 times;

[0039] 2.3 Remove the supernatant, add 1mL leukocyte lysate, vortex and mix evenly, divide evenly into two 1.5mL centrifuge tubes, and store one tube at -80°C for later use;

[0040] 2.4 Add 20 μL proteinase K (10 mg / mL) to another tube, gently invert 8 times to mix, incubate overnight at 56°C (until there is no visible suspension), and invert occasionally to mix;

[0041] 2.5 Add an equal volume of saturated phenol (500 μL), invert and mix well, centrifuge at 12,000 rpm for 10 minute...

Embodiment 3

[0047] Example 3: PCR amplification of exon 9 of PIK3CA gene and sequencing

[0048] 1. Experimental materials

[0049] 2×PfuMix, DNA template extracted in Examples 1 and 2, deionized water, forward and reverse primers for amplifying exon 9 of PIK3CA gene (see Table 1 for details), PCR machine, 1% agarose gel ,Electrophoresis.

[0050] Table 1

[0051]

[0052] 2. Experimental method

[0053] 2.1 Select DNA samples with better quality in Examples 1 and 2, including 88 cases of breast cancer tissue DNA, 19 cases of breast cancer paracancerous tissue DNA, and 22 cases of normal human blood sample DNA;

[0054] 2.2 Configure the PCR reaction system in the following table:

[0055] Table 2

[0056] .

[0057] 2.3 Reaction conditions: ①94°C, 5min pre-denaturation; ②94°C, 30s denaturation; ③55°C, 30s annealing; ④72°C, 35s extension; cycle ② to ④35 times; ⑤72°C, 5min;

[0058] 2.4 After the PCR reaction is completed, spread 1% gel for detection and send it to Sangon Bio...

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Abstract

The invention discloses a breast cancer PIK3CA mutant gene and an application thereof. The breast cancer PIK3CA mutant gene has the c.1658_1659GT>C mutation site, and the nucleotide sequence of the breast cancer PIK3CA mutant gene is shown as SEQ ID NO:1. According to the invention, through a direct sequencing method, PIK3CA gene mutation in 88 breast cancer tissues, 19 breast cancer para-carcinoma tissues and 22 normal human blood samples is detected, and for the first time, it is found that the PIK3CA gene S553fs*7 mutation is as high as 44.3% in the breast cancer tissues, while no mutation occurs in the breast cancer para-carcinoma tissues and the normal human blood samples; it prompts that the mutation can be used for assisting the early diagnosis of breast cancer; and therefore, the breast cancer PIK3CA mutant gene has great significances for preventing and treating occurrence and development of breast cancer in clinic.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a PIK3CA mutation gene in breast cancer and its application in auxiliary preparation of breast cancer diagnostic reagents. Background technique [0002] Breast cancer, the nightmare of all women and families. The Global Cancer Epidemiological Statistics (GLOBOCAN) shows that about 1.3 million women suffer from breast cancer each year, and 458,000 die from breast cancer. Whether in developed or developing countries, breast cancer is currently the most common disease faced by women all over the world. Malignant tumors are also an important cause of death in women. Since the 1990s, the incidence rate of breast cancer in China has increased more than twice that of the world, and the number of new breast cancer cases and deaths in China accounted for 12.2% and 9.6% of the world's annual breast cancer incidence respectively1. At present, breast cancer is the cancer wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12Q1/68
Inventor 盛苗苗罗瑛周小龙唐文如王芳赵月光李珊珊
Owner KUNMING UNIV OF SCI & TECH
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