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Specific primers and liquid-phase chip for chromosome 13q22 region SNP detection

A chromosome and detection solution technology, applied in the field of molecular biology, can solve the problems such as the detection throughput can not meet the clinical needs, the false positive rate is high, and the samples are easily contaminated, so as to avoid uncertain factors, good detection specificity, and detection consistent effect

Inactive Publication Date: 2014-11-26
SUREXAM BIO TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0009] At present, there is no product on the market to detect the gene polymorphism of the chromosome 13q22 segment at home and abroad, and there are very few reports on the gene polymorphism, and most of the reports are still in the experimental research stage. The existing detection technologies are mainly based on On the basis of PCR technology, such as direct sequencing method, PCR-RFLP technology, traditional solid-phase chip and other methods, the traditional solid-phase chip has the defects of high price and poor repeatability, while PCR-based sequencing method and RFLP technology have the disadvantages of sample contamination , the disadvantage of high false positive rate, and at the same time, due to the limitation of detection throughput, it cannot meet the clinical needs

Method used

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  • Specific primers and liquid-phase chip for chromosome 13q22 region SNP detection
  • Specific primers and liquid-phase chip for chromosome 13q22 region SNP detection
  • Specific primers and liquid-phase chip for chromosome 13q22 region SNP detection

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Effect test

Embodiment 1

[0025] Embodiment 1 Chromosome 13q22 segment SNP detection liquid chip mainly includes:

[0026] 1. ASPE Primers

[0027] Specific primer sequences were designed for the wild-type and mutant types of two common genotypes A94G and G288A on chromosome 13q22. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0028] Table 1 ASPE primer sequence (specific primer sequence) of chromosome 13q22 segment

[0029]

[0030]

[0031] Table 2 ASPE primer sequence (tag sequence) of chromosome 13q22 segment

[0032]

[0033] Each ASPE primer includes two parts, the 5' end is the specific tag sequence for the anti-tag sequence on the corresponding microsphere (as shown in the above table 2), and the 3' end is the mutant or wild type specific primer fragment ( as shown in Table 1 above). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was...

Embodiment 2

[0045] Example 2 Using the chromosome 13q22 segment SNP detection liquid chip described in Example 1 to detect the sample, the formulations of the various solutions are as follows:

[0046] 50mM MES buffer (pH5.0) formula (250ml):

[0047]

[0048] 2×Tm hybridization buffer

[0049]

[0050] Store at 4°C after filtration.

[0051] ExoSAP-IT kit was purchased from US USB Company.

[0052] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0053] 1. Sample DNA extraction:

[0054] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0055] 2. PCR amplification of samples to be tested

[0056] Design 2 pairs of primers, multiplex PCR to amplify 2 target sequences containing two common genotypes A94G and G288A in the chromosome 13q22 segment in one step, the product sizes are 282bp and 448bp, respectively, primer sequences (SEQ ID NO.17-20) See Table 4 above.

[005...

Embodiment 3

[0102] Example 3 Detection of Chromosome 13q22 SNP Site by Liquid Chip with Different ASPE Primers

[0103] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0104] Taking the SNP detection liquid chip at the A94G and G288A sites of chromosome 13q22 as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and SNP type of A94G and G288A, and the Tag5 sequence at the 5' end of the ASPE primer was selected. From SEQ ID NO.5-SEQ ID NO.10, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.11-SEQ ID NO.16. The specific design is shown in the following table (Table 9). The synthesis of ASPE primers, microspheres coated with anti i-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0105] Table 9 Design of liquid phase chip preparation

...

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Abstract

The invention provides a liquid-phase chip for chromosome 13q22 region SNP detection. The liquid-phase chip mainly comprises: ASPE primers composed of tag sequences of 5' end and sequences of specific primers for SNP sites of target genes of 3' end, wherein the sequences of the specific primers are SEQ ID No.1 and SEQ ID No.2 for A94G site, and / or SEQ ID No.3 and SEQ ID No.4 for G288A site; microballoons coated by anti-tag sequences; and amplimers. The liquid-phase chip for chromosome 13q22 region SNP detection and a sequencing method reach 100% agreement in detection results, and parallel detection of wide type and SNP type of a plurality of SNP sites can be realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a specific primer for detecting chromosome 13q22 section SNP and a liquid phase chip. Background technique [0002] The target detection mutation site of the present invention is located in the non-genic region of chromosome 13q22.1. In 2010, Petersen et al. revealed through genome-wide association study (GWAS) on Nat Genet that the target detection mutation site of the present invention It is closely related to the occurrence and development of various cancers. At present, the mutation site and the susceptibility risk of pancreatic cancer have become a research hotspot. [0003] The chromosome 13q22 section SNP site of target detection of the present invention, as shown in the table: [0004] [0005] SEQ ID NO.25 (Note: The bases in the box are the target detection SNP sites) SNP site: A94G [0006] ACTGCTCCAAGAGGTTATCTGTGTCCTGCCAGGAC...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C40B40/06
Inventor 许嘉森刘志明
Owner SUREXAM BIO TECH
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