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Mononucleotide polymorphism detecting process based on Linker-PCR and magnetic nano-particles

A single nucleotide polymorphism, magnetic nanoparticle technology, applied in the field of genetic testing, to achieve the effect of automatic detection and low cost

Inactive Publication Date: 2008-11-19
何农跃 +1
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Problems solved by technology

However, the existing SNP detection methods are difficult to meet this demand. Therefore, it is necessary to explore and establish a multi-site SNP detection method that is efficient, low-cost, high-throughput and suitable for clinical applications.

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  • Mononucleotide polymorphism detecting process based on Linker-PCR and magnetic nano-particles

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Embodiment approach

[0028] 1) Select some important functional SNP sites, and design two wild and mutant detection probes respectively labeled Cy3 and Cy5 for each site to be tested. The site to be detected is located in the middle of the probe sequence and each probe’s One end is labeled.

[0029] 2) Restriction endonuclease MseI digestion: Genomic DNA was digested with a sufficient amount of restriction endonuclease MseI at 60° C. overnight. This endonuclease (recognition sequence is T*TAA) digests genomic DNA into short fragments, the fragment size is between 200-2000 bases. Since the recognition sequence of the enzyme avoids most of the SNP sites, the functional SNP sites of each gene remain basically intact after being digested by the enzyme. Digestion products were purified by a purification kit.

[0030] 3) Prepare Linker. Linker is composed of two oligonucleotide chains L1 and L2, the sequence of which is L1: 5'-Biotin-AGG CAA CTG TGC TAT CCG AGGGAT-3' and L2: 5'-TAA TCC CTC GGA-3'. T...

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Abstract

The invention discloses a method for detecting single nucleotide polymorphism (SNP) based on linker polymerase chain reaction (Linker-PCR) and magnetic nano particles. The method is characterized in that: whole genome amplification of a sample is carried out by means of Linker-PCR; a biotin-labeled amplification target sequence is fixed on avidin-labeled magnetic nano particles; and sample typing is realized through adopting a method for hybridizing with an allele specific probe. The method makes use of Linker-PCR to realize the synchronous detection and typing of multiple sites; moreover, through taking the magnetic nano particles as hybrid vectors, the method can avoid the disadvantages of high cost, time consumption and labor waste caused by target sequence purification and concentration; therefore, the method has the advantages of multiple sites, low cost, quickness and simplicity and convenience.

Description

Technical field [0001] The present invention relates to a kind of gene detection technique using linker polymerase chain reaction (Linker-PCR) and magnetic nano particle, especially a kind of detection method of multi-site single nucleotide polymorphism (SNP) Background technique [0002] With the completion of the human genome sequencing work, the DNA sequence of the human genome has been preliminarily determined and analyzed. Existing research results show that the human genome contains about 30,000 to 40,000 protein-coding genes, providing a comprehensive and in-depth understanding of individuals and groups. Variations or polymorphisms among genomes are possible and increasingly important. The genetic polymorphism in the human genome is mostly manifested in repetitive sequences, especially short tandem repeat sequences, such as small satellite DNA and microsatellite DNA, and their polymorphism is mainly based on the variation of the repeat sequence copy number. Another, ...

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 何农跃李松刘洪娜陆祖宏王志飞汤建新
Owner 何农跃
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