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Design method of oligonucleotide probe and application thereof

A technology of oligonucleotide probes and design methods, which is applied in the directions of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. , increase the synthesis cost and other problems, to achieve the effect of reducing the screening workload, strong discrimination ability, and excellent specificity

Active Publication Date: 2013-07-10
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] Embodiment 1 detects the probe design of the gene chip of MDRI gene SNPs

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

[0036] 1 CTCTGGCTTC GACGGGGGAC TAGAGGTTAG TCTCACCTCC AGCGCGCCTG AGGCTCATGC

[0037] 61 ATTTGGCTAA TGAGCTGCGGTTTCTCTTCAGGT 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, is the site of mutation, that is, C is the wild type (Normal), and T is the mutant type (Mutation). When designing the probe, mutation sites and artificial insertio...

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 gene chip on a slide carrier to detect MDRI gene SNPs, and compared with the gene chip prepared by the probe reported in the literature.

[0045] The multi-drug resistance gene (MDRI) is located on human chromosome 7 q21.1, and its protein product p-GP is an important member of the ABC transport system and belongs to the ABCB1 family. The expression level of MDRI gene can be used as a reference index to predict the effect of chemotherapy, and it has become a feasible clinical detection method to predict the chemotherapy effect of cancer patients by detecting the polymorphism of MDRI gene. The PCR primer sequences used were:

[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 ...

Embodiment 3

[0058] Example 3 Design of probes for gene chip for detection of beta thalassemia gene mutation

[0059] The human β-globin gene (β-Globin) cluster is located at 11p15.5. The occurrence of β-thalassemia (abbreviated as β-thalassemia) is mainly due to point mutations of 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, is the mutation site, that is, the wild type (Normal) is G, and the mutant type (Mutation) is A. Wh...

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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 invention relates to a method for designing an oligonucleotide probe. The oligonucleotide probe designed by the method can effectively distinguish single nucleotide polymorphisms. The method for designing oligonucleotide probes of the present invention can be applied to all fields of probe design including the preparation of gene chips. Background technique [0002] Single nucleotide polymorphism (single nucleotide polymorphism, SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. It is the most common type of heritable variation in humans, accounting for more than 90% of all known polymorphisms. SNPs exist widely 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 fields in the future: (1) genetic linkage analysis...

Claims

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Application Information

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