Method for detecting hereditary thoracic aortic aneurysm and dissecting associated gene mutation

A gene and connection detection technology, applied in the field of detection of genetic mutations related to hereditary thoracic aortic aneurysm and dissection, can solve the problems of inability to achieve high throughput, affect detection results, and complicated steps, and meet clinical detection requirements and avoid pollution , the effect of simple steps

Inactive Publication Date: 2012-07-25
姬云
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the characteristics and difficulties of the classical multiplex oligonucleotide ligation detection method lie in the design of probes.
Generally, the M13 phage derivatization method is used to prepare long probes, but the steps are complicated and time-consuming
For capillary electrophoresis detection, electrophoresis conditions, pH buffer, etc. will affect the detection results; high-throughput cannot be achieved

Method used

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  • Method for detecting hereditary thoracic aortic aneurysm and dissecting associated gene mutation
  • Method for detecting hereditary thoracic aortic aneurysm and dissecting associated gene mutation
  • Method for detecting hereditary thoracic aortic aneurysm and dissecting associated gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Extract the DNA of the sample to be tested according to conventional methods

[0044] Take the sample to be tested (including whole blood, plasma, serum), refer to the instructions of the Kangwei Century Blood Genome Small Amount Extraction Kit to extract the DNA in the sample. The detailed steps are as follows:

[0045] 1) Add 20μl Protease to the bottom of a 1.5ml centrifuge tube.

[0046] 2) Add 200μl sample.

[0047] 3) Add 200μl Buffer GL to the sample and vortex for 15 seconds. Add 20ul DNase-free RnaseA.

[0048] 4) Incubate at 56°C for 10 minutes.

[0049] 5) Add 200μl of absolute ethanol and vortex for 15 seconds. After mixing, centrifuge briefly to concentrate the liquid on the tube wall and lid to the bottom of the tube.

[0050] 6) Put the Spin Column DM to be used into the Collection Tube, carefully transfer the solution obtained in step 5) into the Spin Column DM, and avoid adding it to the edge of the Spin Column DM, close the tube cap, 8,000rpm (6000xg) ...

Embodiment 2

[0057] Example 2: The DNA sample is denatured and hybridized with the probe, the probe is connected, and the connected probe sequence is amplified by PCR

[0058] 1) Configure the reaction system. Since multiple connection and amplification reactions are carried out in a single tube, add the DNA sample and probe to be tested into the reaction tube, and then add the necessary reagents for the connection reaction and the PCR amplification reaction to the tube. Each 10μl reaction system includes: 0.8μl DNA ligase (5U / μl), 1μl long probe mixed solution (50nM), 1μl short probe mixed solution (50nM), 1μl Buffer II (10×), 0.8μl of 4 kinds of dNTPs mixed solution (2.5mM), 1.6μl magnesium chloride (25mM), 0.1μl DNA polymerase, 0.1μl nicotinamide adenine dinucleotide (50mM), 0.2μl biological Prime-labeled forward universal primer (10mM), 0.2μl reverse universal primer (10mM), 1μl DNA template extracted according to Example 1, and finally add sterile double-distilled water to the entire rea...

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Abstract

The invention relates to a method for detecting hereditary thoracic aortic aneurysm and dissecting associated gene mutation, in particular to a method for detecting TGFBR1, TGFBR2 and ACTA2 gene mutations by using multiple oligonucleotide connection detection probes and liquid-phase chips. The method comprises the following steps: (a) designing and manufacturing a group of multiple oligonucleotide connection detection probes for 13 mutation sites of the TGFBR1, TGFBR2 and ACTA2 genes, wherein the multiple oligonucleotide connection detection probes have the nucleotide sequences of SEQ ID No. 1 and SEQ ID No. 26; (b) carrying out DNA (deoxyribonucleic acid) sample denaturation, probe hybridization and ligation reaction; (c) carrying out PCR (polymerase chain reaction) amplification to the sequences of the multiple oligonucleotide connection detection probes by using universal primers; and (d) detecting the products of amplification by using the liquid-phase chips. The technology can realize the single-tube reaction detection to dozens of mutation sites, is high in throughput and low in cost, and is easy to operate.

Description

Technical field [0001] The invention belongs to the field of molecular biology, relates to medical diagnosis and biotechnology, and relates to a method for detecting genetic mutations related to hereditary thoracic aortic aneurysm and dissection, in particular to detecting hereditary thoracic aortic aneurysm and dissection related gene TGFBR1 , TGFBR2 and ACTA2 mutation multiple oligonucleotide ligation detection probe combined with liquid chip detection method. Background technique [0002] Thoracic Aortic Aneurysms is a pathological dilation of a certain section of the thoracic aorta. According to pathological anatomy, it can be divided into true and false aneurysms. The incidence rate increases with age, reaching a peak at the age of 50 to 70, with more men than women. Thoracic aortic aneurysm is not a real tumor, but one or more parts of the aorta pulled out due to various reasons, and the "tumor-like" changes that appear are called aortic aneurysms. Thoracic aortic aneury...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 姬云
Owner 姬云
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