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Method for carrying out gene mutation detection on cell-free system by HRM technology

A non-cellular and systemic technology, applied in the fields of biotechnology and medicine, can solve the problems of tissue sample sampling troubles, etc., and achieve convenient and fast effects of real-time monitoring, high sensitivity, and accuracy

Inactive Publication Date: 2011-01-26
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] The main purpose of the present invention is to provide a method for detecting gene mutations in non-cellular systems using HRM technology, using HRM technology to detect trace amounts of free DNA in non-cellular systems, so as to amplify the sample source of gene detection and achieve more accurate and sensitive Genetic testing, which solves the troublesome status of tissue sample sampling in existing testing methods

Method used

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  • Method for carrying out gene mutation detection on cell-free system by HRM technology
  • Method for carrying out gene mutation detection on cell-free system by HRM technology
  • Method for carrying out gene mutation detection on cell-free system by HRM technology

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Experimental program
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Effect test

Embodiment 1

[0116] Verification and screening of primers in Example 1, taking KRAS as an example

[0117] Primers were designed according to the sequences of the second exon and the third exon of the KRAS gene. The different sequence lengths of the primers were amplified and compared, and the primers with good specificity were screened. The specificity results of the PCR amplification of the primers with the size of the primer fragments are as follows and figure 1 Shown:

[0118] The primers of table 1 primer fragment size carry out the specificity result of PCR amplification

[0119] Primer large

small (bp)

5-9

13-16

18-23

24-28

30-37

39-45

specificity

very specific

Difference

poor specificity

good specificity

specificity

it is good

poor specificity

specificity

very poor

[0120] According to the above table and figure 1 As shown, the f...

Embodiment 2

[0122] The extraction of embodiment 2 negative control DNA

[0123] sampling:

[0124] 1. Draw 2.5ml of normal human peripheral blood into a sodium citrate (1:9) anticoagulant tube.

[0125] 2. Centrifuge the blood in the anticoagulant tube at 8000rpm for 5 minutes to separate plasma and cells.

[0126] 3. Carefully transfer the plasma into another sterile tube, be careful not to suck white blood cells, and store it at -20°C (1 week).

[0127] The extraction of DNA was carried out according to the following steps: take 2ml of mixed anticoagulated whole blood from a normal person, and then use the TIANamp Genomic DNA Kit (TIANGEN BIOTECH CO., LTD) Genomic DNA Extraction Kit to extract DNA according to the operating instructions of the kit. DNA concentration was quantified using a Nano 1000 quantifier.

[0128]Negative control DNA can be considered qualified when it meets the following conditions: 1. OD value A260 / A230: 2.0-2.2; A260 / A280: 1.8-2.0; 2. The main band of the ele...

Embodiment 3

[0129] Example 3 Extraction of Tumor Tissue DNA

[0130] The sample collection is carried out according to the following steps: Take 50-100 mg of fresh tissue block and wash it with PBS or normal saline. The tissue used must be cut to a thickness of less than 0.5 cm, put it into a cryopreservation tube with a volume of 1.5 ml RNAlater, and mix well (Complete within 30 minutes). Store at room temperature for 1-2 hours, freeze overnight at 4°C, and transfer to -20°C for long-term storage the next day.

[0131] DNA extraction was carried out according to the following steps: use TIANamp Genomic DNA Kit (TIANGENBIOTECH CO., LTD) Genomic DNA Extraction Kit, and follow the instructions of the kit to extract DNA. Then carry out DNA purification and detection. If the OD value of the extracted DNA is not within the standard range of A260 / A230: 2.0-2.2; A260 / A280: 1.8-2.0, a DNA purification step is required.

[0132] DNA purification was carried out as follows: add an equal volume o...

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Abstract

The invention discloses a method for carrying out gene mutation detection on a cell-free system by an HRM technology. The invention is characterized in that the method comprises the following steps: (1) designing and selecting a plurality of primer pairs containing continuous nucleotide sequences formed by at least 15 continuous nucleotides in the targeted region of a target gene; (2) extracting DNA from the cell-free system, and utilizing the plurality of primer pairs screened in advance to carry out specific amplification on the targeted sequence of the target gene; and (3) carrying out mutational site detection on the amplified gene sequence according to a high-resolution fusion curve method. The method provides more extensive selectable samples for the detection and the analysis on related diseases by the cell-free system application technical research and obviously reduces invasive harm of the diseases on patients.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to the rapid detection of genes related to the intermediate information of tumor diagnosis, and the replacement of required detection samples. The rapid detection of genes is realized by detecting trace amounts of DNA in non-cellular systems. Background technique [0002] High-resolution melting analysis (high-resolution melting analysis, HRM) is a new genetic analysis method for mutation scanning and genotyping that has emerged abroad in recent years. Based on efficient and robust PCR technology, HRM is not limited by the site and type of mutated bases, and does not require sequence-specific probes. After PCR, high-resolution melting can be run directly to complete the analysis of sample mutations and single nucleotide polymorphisms. -Analysis of SNP, methylation, HLA matching, etc. The main principle of HRM is to analyze the sample according to the length of the DNA seq...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 王弢秦勇陈菲时凯
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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