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Design method of oligonucleotide probe and application thereof

A technology for oligonucleotide probes and design methods, which can be used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. The increase of density chip preparation and other problems can achieve the effect of strong discrimination ability, excellent specificity, and reduced screening workload.

Active Publication Date: 2011-06-22
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One disadvantage of this method is that blocking the base requires a special reaction, which increases the cost of synthesis, and the corresponding service cannot be completed in commercial synthesis companies, so it cannot be effectively promoted.
For example, S Choi et al. (S Choi, et al.US PatentApp.11 / 335,229) disclose a method of hurdle DNA and artificially mismatched probe (hurdle DNA and artificially mismatched probe), which requires two probes to To realize the detection of one site, since usually only one probe can detect one site, the number of probes must be doubled, which undoubtedly adds great difficulties to the sampling of probes and the preparation of high-density chips. As far as possible, one probe can effectively detect one site, which is still a consideration in the actual application process

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  • Design method of oligonucleotide probe and application thereof
  • Design method of oligonucleotide probe and application thereof
  • Design method of oligonucleotide probe and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Probe design of gene chip for detecting MDRI gene SNPs

[0035] The multi-drug resistance gene (MDRI) is located on human chromosome 7 q21.1, taking C-4T as an example to illustrate the probe design method. The following is the sequence of MDRIC-4T site (LOCUS: AY910577):

[0036] 1 CTCTGGCTTC GACGGGGGAC TAGAGGTTAG TCTCACCTCC AGCGCGCCTG AGGCTCATGC

[0037] 61 ATTTGGCTAA TGAGCTGCGG TTTCTCTTCA GGT GGGATG GATCTTGAAG GGGACCGCAA

[0038] 121 TGGAGGAGCA AAGAAGAAGA ACTTTTTTAA ACTGAACAAT AAAAGGTAAC TAGCTTGTTT

[0039] 181 CATTTTCATA GTTTACATAG TTGCGAGATT TGAGTAATTT ATTTCTAGCC TCCAGCTCTG

[0040] 241 AAATAAATGA CATGTTGTTG TTTTTAATTA TTTTTAAGAA ACGCAAGCTA GCCTTTGGAA

[0041] 301 TCAATATCCC TGCTTAGAGC AGAAGTTTGT TGGCTGAGTG GAGCACAGCA TATGCATTTT

[0042] In the above sequence, It is the site where the mutation occurs, that is, the wild type (Normal) is C, and the mutant type (Mutation) is T. During probe design, mutation sites and artificial insertion sites are distributed at both...

Embodiment 2

[0043] Example 2 Detection of C-4T and A61G sites of MDRI gene by gene chip of slide carrier

[0044] The probe designed in Example 1 was used to prepare a slide carrier gene chip to detect MDRI gene SNPs, and compared with the gene chip prepared by the probe reported in the literature.

[0045] Multi-drug resistance gene (MDRI) is located on human chromosome 7 q21.1. Its protein product p-GP is an important member of the ABC transport system and belongs to the ABCB1 family. MDRI gene expression level can be used as a reference indicator for predicting the effect of chemotherapy. By detecting MDRI gene polymorphisms to predict the efficacy of chemotherapy in cancer patients has become a feasible clinical detection method. The PCR primer sequences used are:

[0046] 1F: 5'TTGGCTAATGAGCTGCGGTTTCTC 3'(SEQ ID NO: 25);

[0047] 1R: 5'cy3-GATTCCAAAGGCAGCTTGCGTTTC 3'(SEQ ID NO: 26).

[0048] The amplified fragment of the above sequence is 240bp, and the two SNPs sites C-4T and A61G of exon 2...

Embodiment 3

[0058] Example 3 Probe design of gene chip for detecting β-thalassemia gene mutation

[0059] The human β-Globin gene (β-Globin) cluster is located at 11p15.5. Beta thalassemia (abbreviated as beta thalassaemia) occurs mainly due to point mutations in genes, and a few are gene deletions. The following is the sequence of β-Globin CD26 (G→A) (LOCUS: NC 000011)

[0060] 1 TGACTCCTGA GGAGAAGTCT GCCGTTACTG CCCTGTGGGG CAAGGTGAAC GTGGATGAAG

[0061] 61 TTGGTGGT A GGCCCTGGGC AGGTTGGTAT CAAGGTTACA AGACAGGTTT AAGGAGACCA

[0062] 121 ATAGAAACTG GGCATGTGGA GACAGAGAAG ACTCTTGGGT TTCTGATAGG CACTGACTCT

[0063] 181 CTCTGCCTAT TGGTCTATTT TCCCACCCTT AGGCTGCTGG TGGTCTACCC TTGGACCCAG

[0064] 241 AGGTTCTTTG AGTCCTTTGG GGATCTGTCC ACTCCTGATG CTGTTATGGG CAACCCTAAG

[0065] 301 GTGAAGGCTC ATGGCAAGAA AGTGCTCGGT GCCTTTAGTG ATGGCCTGGC TCACCTGGAC

[0066] In the above sequence, It is the site where the mutation occurs, that is, the wild type (Normal) is G, and the mutant type (Mutation) is A. During probe desig...

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Abstract

The invention relates to a design method of an oligonucleotide probe, and the oligonucleotide probe designed by the method can effectively distinguish the mononucleotide polymorphism. The design method of an oligonucleotide probe of the invention is applicable to all probe design fields including the preparation of gene chips. The method does not use base analogues, but uses A / T / G / C and adopts a way of manual addition or reduction of bases. Based on the detection effects, the probe designed by the method of the invention is superior to traditional probes in specificity, and no significant difference exists between the two probes in sensitivity.

Description

Technical field [0001] The present invention relates to a method for designing oligonucleotide probes. The oligonucleotide probes designed by the method can effectively distinguish single nucleotide polymorphisms. The oligonucleotide probe design method of the present invention can be applied to all probe design fields including the preparation of gene chips. Background technique [0002] Single nucleotide polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. It is the most common type of human heritable variation, accounting for more than 90% of all known polymorphisms. SNPs are widespread in the human genome, with an average of 1 in every 500-1000 base pairs, and it is estimated that the total number can reach 3 million or more. [0003] It is expected that SNP will play an important role in the following areas in the future: (1) Perform genetic linkage analysis and association analysis of simple and co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
Inventor 程钢林炳生张帆陈华云
Owner DAAN GENE CO LTD
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