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Method for detecting mutation of related genes of genetic long QT syndrome

A gene and ligation detection technology, applied in the field of molecular biology, can solve the problems of inability to achieve high throughput, affect the detection results, complex steps, etc., to improve detection accuracy and stability, avoid laboratory contamination, and simple steps. Effect

Inactive Publication Date: 2012-07-25
苏州科贝生物技术有限公司
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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 mutation of related genes of genetic long QT syndrome
  • Method for detecting mutation of related genes of genetic long QT syndrome
  • Method for detecting mutation of related genes of genetic long QT syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, extract the DNA of the sample to be tested according to conventional methods

[0043] Take the sample to be tested (including whole blood, plasma, and serum), and extract the DNA in the sample according to the instructions of the Kangwei Century Blood Genome Small Extraction Kit. The detailed steps are as follows:

[0044] 1) Add 20μl Protease to the bottom of a 1.5ml centrifuge tube.

[0045] 2) Add 200μl sample

[0046] 3) Add 200 μl Buffer GL to the sample, and vortex for 15 seconds. Add 20ul DNase-free RNaseA.

[0047] 4) Incubate at 56°C for 10 minutes.

[0048] 5) Add 200 μl of absolute ethanol, vortex for 15 seconds, centrifuge briefly after mixing, so that the liquid on the tube wall and the cap is concentrated at the bottom of the tube.

[0049] 6) Put the ready-to-use Spin Column DM into the Collection Tube, carefully transfer the solution obtained in step 5) into the Spin Column DM, and avoid adding to the edge of the Spin Column DM, cover t...

Embodiment 2

[0056] Example 2, DNA sample denaturation and probe hybridization, connecting the probes, and performing PCR amplification on the connected probe sequences

[0057] 1) Configure the reaction system. Since multiple ligation 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 required reagents for the ligation reaction and PCR amplification reaction into 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 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 Primer-labeled forward universal primer (10 mM), 0.2 μl reverse universal primer (10 mM), 1 μl DNA template extracted according to Example 1, and finally add sterile double distilled water until the entire...

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Abstract

The invention relates to a method for detecting mutation of related genes of an genetic long QT syndrome, in particular relates to a method for detecting mutation of related genes, including KCNJ2 and CAV3, of the genetic long QT syndrome by combining a multi-oligonucleotide link detection probe with a liquid phase chip. The method comprises the following steps of: a) designing and preparing the multi-oligonucleotide link detection probe, wherein a probe sequence for mutation of the gene KCNJ2 is SEQ ID No.1-SEQ ID No.32, and the probe sequence for the mutation of the gene CAV3 is SEQ ID No.33-SEQ ID No.40; b) carrying out DNA sample denaturation and probe hybridization, and carrying out link reaction; c) carrying out PCR (polymerase chain reaction) amplification on the probe sequences by adopting a general primer; and d) detecting amplification products by utilizing the liquid phase chip. The technology can realize single-tube reaction detection on dozens of mutation sites at most, flux is high, operation is easy and cost is low.

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 mutations of genes related to hereditary long QT syndrome, in particular to a method for detecting mutations of genes KCNJ2 and CAV3 related to hereditary long QT syndrome A method for combining multiple oligonucleotides with detection probes combined with a liquid phase chip. Background technique [0002] On the electrocardiogram, when the normal heart rate is 60-100 beats / min, the QT interval is generally 320-440 milliseconds, and when the QT interval exceeds 440 milliseconds, it is considered to be prolonged QT interval (LQT). Long QT syndrome (LQTs) refers to a group of syndromes with prolonged QT interval on electrocardiogram, ventricular arrhythmia, syncope and sudden death, which may be accompanied by congenital deafness. LQTs can be either congenital or acquired. We call a class of LQTs with obvious fa...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 侯青姬云张泓
Owner 苏州科贝生物技术有限公司
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