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Mononucleotide polymorphism detecting method based on dU or dI oligonucleotide probe

A single nucleotide polymorphism and oligonucleotide probe technology, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., to achieve the effect of large operation throughput, easy operation and stable results

Inactive Publication Date: 2010-11-17
TAIHU RUIJING BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recent studies have shown that pfu DNA polymerase specifically tightly binds deoxyuridine dU nucleotides and dI nucleotides in DNA, and stagnates at dU or dI, causing DNA synthesis reactions to fail to proceed smoothly

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0019] Example The determination of the 21st nucleotide species of Chlamydia endonuclease IV gene

[0020] In this embodiment, the SNP site to be tested is the 21st nucleotide species of the Chlamydia endonuclease IV gene, the thermostable mutS protein is TthmutS, the reporter DNA is the ampicillin resistance gene, and the reporter DNA template molecule is the pUC18 plasmid , the detection probe is a dU or dI oligonucleotide probe, and this embodiment is a dU oligonucleotide probe. The specific implementation steps are as follows:

[0021] The first step is to design and synthesize a dU oligonucleotide probe for determining the 21st nucleotide type of the Chlamydia endonuclease IV gene. The four oligonucleotide probes were CpendoIV-21-T, CpendoIV-21-A, CpendoIV-21-C, and CpendoIV-21-G. The base sequences of the four dU oligonucleotide probes are as follows:

[0022] CpendoIV-21-T: 5'agtaagggaa tggatggagg aggaagtact xtcat

[0023] CpendoIV-21-A: 5'agtaagggaa tggaaggagg agga...

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Abstract

The invention relates to a detection method for the polymorphism of mononucleotide based on a dU or dI oligonucleotide probe. The dU or dI oligonucleotide probe has the sequence characteristics that 30 to 40 nucleotides are provided in the total length, a nucleotide at the 15-25 position at the fifth end is paired with an SNP locus to be detected, a nucleotide at the 1-15 position at the third end is dU or dI, the dU or dI is 5 to 15 nucleotides away from the SNP locus to be detected, and the entire oligonucleotide probe is paired with the SNP locus to be detected and the nucleotides on the two sides thereof. The reaction components comprise the dU or dI oligonucleotide probe, monochain DNA to be detected, heat stable mutS albumen, report DNA template molecules, report DNA idiosyncratic primer, dNTPs, pfuDNA polymerase and the reaction buffer solution thereof. After the dU or dI oligonucleotide probe and the monochain DNA to be detected are hybridized, if mispaired basic groups are formed at the SNP locus, a PCR can amplify the report DNA, and if correctly paired basic groups are formed at the SNP locus, the report DNA cannot be amplified. According to the DNA amplification result, the types of the basic groups at the SNP locus to be detected can be presumed.

Description

technical field [0001] The present invention relates to a kind of new single nucleotide polymorphism (single nucleotide polymorphism, SNP) detection method, relate in particular to a kind of SNP detection method based on dU or dI oligonucleotide probe, can be used for gene polymorphism Analysis and species typing, etc., belong to the technical field of biological detection. Background technique [0002] As a DNA sequence polymorphism caused by a single nucleotide variation at the genome level, SNP is widely used in genetic disease-causing gene association analysis, linkage disequilibrium analysis, pharmacogenomics, patient personalized treatment, forensic identification, It has important applications in fields such as crop genetics and breeding. In view of the important position of SNP in modern life science, a variety of SNP assay methods have been developed. However, the existing SNP determination method or detection cost is high [BMC Genomics, 2006, 7: 291-291], or the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 刘喜朋刘建华
Owner TAIHU RUIJING BIO TECH