Specific primers and liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of LPL gene

A detection solution and specific technology, applied in the field of molecular biology, can solve the problems of insufficiency and inability to use, and achieve the effects of consistent detection effect, simple steps and low cross-reaction rate.

Active Publication Date: 2012-07-04
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.
Thirdly, both the detection method based on PCR technology (PCR-RFLP) and the allelic difference analysis method based on TaqMan technology have limitations in the detection throughput, and only one mutation can be detected at a time, which cannot meet the needs of practical applications.

Method used

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  • Specific primers and liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of LPL gene
  • Specific primers and liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of LPL gene
  • Specific primers and liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of LPL gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 LPL gene SNP detection liquid chip mainly includes:

[0024] 1. ASPE Primers

[0025] Specific primer sequences were designed for the wild-type and mutant types of the four common genotypes T161G, C 120G, G113A and A178G of the LPL gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0026] The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1LPL gene

[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 100pmol / mL stock solution with 10mmol / LTris Buffer.

[0029] 2. Microspheres coated with anti-tag sequences

[0030] Acc...

Embodiment 2

[0040] Example 2 Detection of samples using LPL gene detection liquid chip

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

[0042] 50mM MES buffer (pH5.0) formulation (250mL):

[0043]

[0044] 2×Tm hybridization buffer

[0045] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5M NaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0046] Store at 4°C after filtration.

[0047] ExoSAP-IT kit was purchased from US USB Company.

[0048] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0049] 1. Sample DNA extraction

[0050] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0051] 2. PCR amplification of samples to be tested

[0052] Four pairs of primers were designed, and multiplex PCR was...

Embodiment 3

[0110] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of LPL gene SNP site

[0111] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0112] Taking the C 120G site mutation detection liquid chip of the LPL gene as an example, the specific primer sequences at the 3' end of the ASPE primer were designed for the wild type and mutant type of C 120G, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO .1-SEQ ID NO.8, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.17-SEQ ID NO.24. 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.

[0113] Table 7 Design of liquid phase chip preparation

[0114]

...

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PUM

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Abstract

The invention discloses specific primers and a liquid-phase chip for SNP (Single Nucleotide Polymorphism) detection of an LPL gene. The liquid-phase chip comprises an ASPE primer, anti-tag sequence coated microspheres, and an amplification primer, wherein the ASPF primer is composed of a 5'-terminal tag sequence and 3'-terminal specific primers for target gene mutation, and the specific primers are respectively SEQ ID NO. 9 and SEQ ID NO. 10 specific for a T161G SNP site, SEQ ID NO. 11 and SEQ ID NO. 12 specific for a C120G SNP site, SEQ ID NO. 13 and SEQ ID NO. 14 specific for a G113A SNP site, and / or SEQ ID NO. 15 and SEQ ID NO. 16 specific for an A178G SNP site. According to the invention, the coincidence rate between the detection result of the liquid-phase chip for SNP detection of the LPL gene and the detection result of a sequencing method is up to 100%, not only is the condition of single site mutation detected, but also the polymorphism conditions of multiple mutant sites can be simultaneously detected in parallel, and the detection effects are consistent.

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 LPL gene SNP detection and a liquid phase chip. Background technique [0002] Lipoprotein lipase (lipoprteinlipase, LPL) is a glycoprotein synthesized and secreted by parenchymal cells such as adipocytes, cardiomyocytes, skeletal muscle cells, breast cells, and macrophages, with a molecular weight of 60ku and a carbohydrate content of 3%-8% . The physiological function of LPL is to catalyze the decomposition of TG in the core of CM and VLDL into fatty acids and monoglycerides for tissue oxidation, energy supply and storage. LPL is also involved in the conversion of apolipoprotein and phospholipid between VLDL and HDL. ApoC II is an essential cofactor for LPL, and its C-terminal amino acids 61-79 can activate LPL. [0003] The LPL gene is located at 8p22, the short arm of the eighth chromosome, and is about 35kb long. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 许嘉森秦会娟朱泽尧孙贤标
Owner SUREXAM BIO TECH
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