Specific primers and liquid phase chip for polymorphic detection of age-related maculopathy susceptibility 2 (ARMS2) gene and high temperature factor A-1 (HTRA1) gene

A technology for gene polymorphism and detection solution, applied in the field of molecular biology, can solve problems such as inability to use and cannot meet practical applications, and achieve consistent detection results, good signal-to-noise ratio, and avoid cross-reaction effects.

Inactive Publication Date: 2012-01-04
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR-RFLP method is based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific fragment is amplified by PCR, and then cut and amplified with a restriction endonuclease. Amplify the product and observe the size of the fragment by electrophoresis. This method is used to detect gene mutations with altered restriction sites, and can directly determine the genotype. However, this method cannot be used for the detection of gene mutations without new restriction sites.
Again, the above methods all have limitations in detection throughput, and can only detect one mutation type at a time, which cannot meet the needs of practical applications.

Method used

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  • Specific primers and liquid phase chip for polymorphic detection of age-related maculopathy susceptibility 2 (ARMS2) gene and high temperature factor A-1 (HTRA1) gene
  • Specific primers and liquid phase chip for polymorphic detection of age-related maculopathy susceptibility 2 (ARMS2) gene and high temperature factor A-1 (HTRA1) gene
  • Specific primers and liquid phase chip for polymorphic detection of age-related maculopathy susceptibility 2 (ARMS2) gene and high temperature factor A-1 (HTRA1) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 ARMS2 and HTRA1 gene polymorphism detection liquid chip mainly includes:

[0025] 1. ASPE Primers

[0026] Specific primer sequences were designed for the wild-type and mutant types of the common mutation site G72T in the ARMS2 gene, and the wild-type and mutant types of the common I / D mutation and C95G / A and G60A mutation sites in the HTRA1 gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0027] The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1 ARMS2 and HTRA1 gene

[0028]

[0029] 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 ...

Embodiment 2

[0042] Example 2 Using the ARMS2 and HTRA1 gene polymorphism detection liquid chip described in Example 1 to detect samples The formulations of the various solutions are as follows:

[0043] 50mM MES buffer (pH5.0) formula (250ml):

[0044]

[0045] 2×Tm hybridization buffer

[0046] Reagent

source

Final concentration

Dosage per 250ml

MTris-HCl, pH 8.0

SigmaT3038

0.2M

50ml

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 samples to be tested

[0053] Four pairs of primers we...

Embodiment 3

[0096] Example 3 Detection of ARMS2 and HTRA1 gene polymorphism sites by liquid chip with different ASPE primers

[0097] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0098] Taking the G72T site mutation of the ARMS2 gene and the G60A mutation detection liquid chip of the HTRA1 gene as examples, the specific primer sequences at the 3' end of the ASPE primers were designed for the wild type and mutant types of G72T and G60A, and the Tag sequence at the 5' end of the ASPE primers It is selected from SEQ ID NO.1-SEQ ID NO.9, and correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.19-SEQ ID NO.27. 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.

[0099] Table ...

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Abstract

The invention relates to a liquid phase chip for the polymorphic detection of an age-related maculopathy susceptibility 2 (ARMS2) gene and a high temperature factor A-1 (HTRA1) gene. The liquid phase chip comprises allele specific primer extension (ASPE) primers consisting of tag sequences at the 5' end and specific primers aiming at a mutant target gene at the 3' end, microspheres which are wrapped with anti-tag sequences and an amplification primer, wherein the specific primers are SEQ ID No. 10 and SEQ ID No. 11 for a G72T mutant locus of the ARMS2 gene, SEQ ID No. 12 to SEQ ID No. 14 for a C95G / A mutant locus of the HTRA1 gene, SEQ ID No. 15 and SEQ ID No. 16 for a G60A mutant locus of the HTRA1 gene and / or SEQ ID No. 17 and SEQ ID No. 18 for the insertion / deletion (I / D) mutation of the HTRA1 gene. The coincidence rate of a detection result of the liquid phase chip and a sequencing method is up to 100 percent, cross reaction can be prevented, and the parallel detection of multiplepolymorphic loci is 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 ARMS2 and HTRA1 gene polymorphisms and a liquid phase chip. Background technique [0002] Age-related macular degeneration (AMD) is a common eye aging disease caused by degenerative changes in the macula, which can gradually destroy central vision and eventually lead to blindness. AMD with soft drusen, pigmentary abnormalities, and geographic atrophy is often referred to as dry AMD, whereas AMD with choroidal neovascularization, retinal pigment epithelial (RPE) detachment, or discoid fibrosis is termed wet or neoplastic Vascular AMD. In recent decades, AMD has become one of the main causes of blindness in western developed countries. With the aging population in my country, the number of retinal diseases such as AMD is also increasing, and its pathogenesis and treatment methods are receiving more and more att...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森郭婧刘志明覃晓霞
Owner SUREXAM BIO TECH
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