Chemiluminescence detection method for copy number polymorphism based on magnetic separation and primer extension

A technology for chemiluminescence detection and copy number polymorphism, which is applied in the field of molecular genetics and can solve problems such as high cost

Active Publication Date: 2012-02-22
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purpose of the present invention is to solve the above-mentioned problem of high cost of obtaining nucleotide sequence copy number information caused by the use of special ligases, and proposes a method for obtaining nucleotide sequence copies based on magnetic separation that can greatly reduce costs. After obtaining the copy number information of the nucleotide sequence, detect chemiluminescence to analyze gene copy number variation, so as to lay the foundation for the promotion of CNVs detection as a routine genetic variation detection project

Method used

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  • Chemiluminescence detection method for copy number polymorphism based on magnetic separation and primer extension
  • Chemiluminescence detection method for copy number polymorphism based on magnetic separation and primer extension
  • Chemiluminescence detection method for copy number polymorphism based on magnetic separation and primer extension

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Acquisition of nucleic acid sequence copy number information using artificially synthesized GAPDH gene fragments as templates

[0043] The experimental process of obtaining the copy number information of the nucleic acid sequence using the artificially synthesized GAPDH gene fragment as a template is as follows:

[0044] 1) Preparation of magnetic particles. Preparation of Fe with a diameter of about 300 nm by solvothermal method 3 o 4 magnetic particles. Weigh 1.35 g of FeCl 3 ·6H 2 O, 3.6 g of sodium acetate (NaAc) and 1.0 g of polyethylene glycol (polyethylene glycol, PEG), dissolved in 40 mL of ethylene glycol, magnetically stirred to dissolve, forming a yellow-brown colloid. Transfer the colloid to a tetrafluoroethylene reactor, seal it, and place it in an oven at 200°C for 8 hours; after the reaction is completed, wash it with ethanol and deionized water, and dry it to obtain Fe 3 o 4 Magnetic particle samples such as figure 2 A show.

[0045] ...

Embodiment 2

[0053] Example 2 Acquisition of nucleic acid sequence copy number information using genomic DNA as a template

[0054] 1) Preparation of magnetic particles. Magnetic particles were prepared as described in Example 1 for use.

[0055] 2) Primer and probe design. The primers and probes for GAPDH, GSTT1 and GSTM1 described in Example 1 were used.

[0056] 3) The product capture method obtains the copy number information of the target nucleic acid fragment in the human genome DNA template. Add 5 μL of genomic DNA template, denature at 98°C, add hybridization buffer and primers, and hybridize, so that the primers are fully combined with the genomic DNA template. The copy number information of the target nucleic acid fragment in the genomic DNA template was obtained by following the reaction system and specific experimental steps described in Example 1. The result is as Figure 4 shown. Lane 2 is the human genome DNA template control, and lane 3 is the primer control. Lane 1 ...

Embodiment 3

[0059] Example 3 Chemiluminescent detection of extension products of GAPDH, GSTT1 and GSTM1

[0060] Examples 1 and 2 demonstrate that the target nucleic acid fragment can be obtained through primer extension reaction using artificially synthesized DNA as a template or genomic DNA as a template, and the following detection work can be performed.

[0061] 1) Preparation of magnetic particles. Magnetic particles were prepared as described in Example 1 for use.

[0062] 2) Primer and probe design. The primers and probes for GAPDH, GSTT1 and GSTM1 described in Example 1 were used.

[0063] 3) Extension formation of biotin-labeled nucleic acid fragments. The specific experimental process is as follows: add 10×Buffer 2 μL, Mg 2+ (25 mM) 2 μL, dNTP (2.5 mM) 1.5 μL (including Biotin-dUTP / dTTP: 4 / 21), primer 2.5 μL, artificially synthesized DNA template 2.5 μL, DNA polymerase 0.5 μL, add deionized water to make up the volume to 20 μL and place the reaction mixture in a PCR machin...

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Abstract

The invention discloses a chemiluminescence detection method for copy number polymorphism based on magnetic separation and primer extension. The method comprises the following steps of: preparing a magnetic medium suitable for acquiring nucleic acid sequence copy number information; designing primers and probes specifically combined with a target nucleic acid sequence; determining a Tm value according to the primer design data, so that the primers are fully combined with a DNA template denatured into a single chain; adding a extension reaction buffer solution, and capturing target nucleic acid fragments to the magnetic medium; performing magnetic separation; and performing chemiluminescence detection on target gene and internal reference gene nucleic acid fragments reflected into the template in the magnetic medium, comparing the difference of the chemiluminescence ratios of the target gene / internal reference gene nucleic acid fragments in a template to be detected and a control template, and thus acquiring the copy number change information of the target genes in the template to be detected relative to the control template.

Description

[0001] technical field [0002] The invention belongs to the field of molecular genetics, and relates to a method for detecting copy number polymorphism with chemiluminescence based on magnetic separation and primer extension. The present invention can be applied to the discovery and identification of genetic abnormalities, in particular, the present invention is particularly applicable to the discovery of microscopic and submicroscopic genome structure variations, including deletions, duplications, and polymorphisms of large segments, so as to quantify and correlate with normal and disease states associated genomic changes. The present invention can also be applied to other gene quantitative studies. [0003] Background technique [0004] Genetic variation is a fundamental characteristic of life and an important indicator of disease. At present, genome-wide association studies (GWAS) based on the third-generation genetic marker - single nucleotide polymorphism (SNP) has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 曾新何农跃柳明
Owner SOUTHEAST UNIV
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