Specific primer and liquid-phase chip for SNP (single nucleotide polymorphism) detection of PIGU (phosphatidylinositol glycan anchor biosynthesis, class U) genes

A detection liquid and specificity technology, which is applied in the field of molecular biology, can solve the problems that the detection flux cannot meet the clinical needs, the false positive rate is high, and the sample is easy to be polluted, so as to achieve accurate and reliable detection results, good detection specificity, The effect of strong expansion

Inactive Publication Date: 2011-09-21
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are almost no products on the market to detect the polymorphism of the PIGU gene at home and abroad, and there are very few reports on the polymorphism of the gene, and most of the reports are still in the stage of experimental research and have not yet been commercialized. The existing detection technologies are mainly based on On the basis of PCR technology, such as direct sequencing method, real-time fluorescence quantitative PCR method, etc., these technologies have the disadvantages of easy sample contamination and high false positive rate, and at the same time, due to the limitation of detection throughput, they cannot meet the clinical needs

Method used

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  • Specific primer and liquid-phase chip for SNP (single nucleotide polymorphism) detection of PIGU (phosphatidylinositol glycan anchor biosynthesis, class U) genes
  • Specific primer and liquid-phase chip for SNP (single nucleotide polymorphism) detection of PIGU (phosphatidylinositol glycan anchor biosynthesis, class U) genes
  • Specific primer and liquid-phase chip for SNP (single nucleotide polymorphism) detection of PIGU (phosphatidylinositol glycan anchor biosynthesis, class U) genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 PIGU gene polymorphism detection liquid chip mainly includes:

[0022] 1. ASPE Primers

[0023] Specific primer sequences were designed for the wild type and mutant type of the common genotype G86A of the PIGU gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0024] Specific primer sequence in table 1 PIGU gene ASPE primer sequence

[0025]

[0026] Tag sequence in table 2 PIGU gene ASPE primer sequence

[0027]

[0028] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.

[0029] 2. Microspheres coa...

Embodiment 2

[0040] Example 2 Application of PIGU gene detection liquid chip to sample detection

[0041] The formula of described various solutions is as follows:

[0042] 50mM MES buffer (pH5.0) formula (250ml):

[0043]

[0044] 2×Tm hybridization buffer

[0045] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

[0046] 5MNaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0047] Store at 4°C after filtration.

[0048] ExoSAP-IT kit was purchased from US USB Company.

[0049] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0050] 1. Sample DNA extraction:

[0051] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0052] 2. PCR amplification of test samples

[0053] The target sequence of the common genotype G86A c...

Embodiment 3

[0095] The liquid phase chip of embodiment 3 different ASPE primers detects the polymorphic site of PIGU gene

[0096] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0097] Taking the PIGU gene G86A site mutation detection liquid chip as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and mutant type of G86A, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.3 - SEQ ID NO.8, correspondingly, the anti-tag sequence coated on the microspheres and complementary to the corresponding tag sequence is selected from SEQ ID NO.9-SEQ ID NO.14. The specific design is shown in the following table (Table 7). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0098] Table 7 Design of liquid phase chip preparation

[0099]

[0100] 2. ...

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Abstract

The invention discloses a liquid-phase chip for SNP (single nucleotide polymorphism) detection of PIGU (phosphatidylinositol glycan anchor biosynthesis, class U) genes, which mainly comprises an ASPE (allele specific primer extension) primer, a microsphere and an amplification primer, wherein the ASPE primer is composed of a tag sequence at a 5' terminal and specific primer sequences aiming at the mutant sites of target genes at a 3' terminal; the specific primer sequences comprise one of wild-type SEQ ID NO.1, SEQ ID NO.18 and SEQ ID NO.19 and one of mutant-type SEQ ID NO.2, SEQ ID NO.20 and SEQ ID NO.21; and the microsphere is coated by an anti-tag sequence. The coinciding rate of the detection result of the liquid-phase chip provided by the invention and the detection result of a sequencing method reaches 100%, and wild-type and mutant-type parallel detection of SNP sites is realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a PIGU gene SNP detection specific primer and a liquid phase chip. Background technique [0002] PIGU (phosphatidylinositol glycan anchor biosynthesis, class U) gene, also known as CDC91L1, is the fifth subunit of GPI transamidase, and the protein encoded by this gene plays an important role in the regulation of cell division. [0003] The PIGU gene mutation site detected by the target of the present invention is as shown in the table: [0004] serial number Content of PIGU site mutation abbreviation 1 The 86th nucleotide of SEQ ID NO.17 undergoes a G→A mutation G86A [0005] At present, there are almost no products on the market to detect the polymorphism of the PIGU gene at home and abroad, and there are very few reports on the polymorphism of the gene, and most of the reports are still ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森何嘉英陈家欣
Owner SUREXAM BIO TECH
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