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Specific primers and liquid-phase chips for mc1r gene mutation detection

A detection solution and chip technology, applied in the field of molecular biology, can solve problems such as difficult to meet practical applications, low degree of automation, high false positive rate, etc., and achieve the effect of avoiding uncertain factors, simple steps, and good detection specificity

Active Publication Date: 2016-08-03
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, MC1R gene mutation detection methods mainly include: Illumina fiber optic bead chip technology, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and fluorescent quantitative PCR technology, although Illumina fiber optic bead chip technology is highly sensitive and Accurate high-throughput detection system, but the degree of automation is low, and manual operations are more, which is difficult to meet the needs of practical applications
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a soft ionization technology that has powerful and mature functions in the detection of protein and other biological macromolecules. However, in the field of nucleic acid detection, due to the particularity of nucleic acid molecules, detection is subject to certain limit
Fluorescent quantitative PCR technology has the characteristics of high sensitivity, strong specificity, and high degree of automation, but it also has the disadvantages of easy sample contamination and high false positive rate, and can only detect one mutation type at a time

Method used

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  • Specific primers and liquid-phase chips for mc1r gene mutation detection
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  • Specific primers and liquid-phase chips for mc1r gene mutation detection

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1 MC1R gene mutation detection liquid chip mainly includes:

[0021] 1. ASPE Primers

[0022] Specific primer sequences were designed for the wild-type and mutant types of the three common genotypes C451T, C478T and G565T of the MC1R gene. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0023] Table 1 ASPE primer sequence of MC1R gene (tag sequence + specific primer sequence)

[0024]

[0025] Each ASPE primer consists of 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 Table 1 above). 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 / LTrisBuffer.

[0026] 2. Microspheres coated with anti-tag sequences

[0027] ...

Embodiment 2

[0037] Example 2 Detection of samples using the MC1R gene mutation detection liquid chip described in Example 1

[0038] The formula of described various solutions is as follows:

[0039] 50mM MES buffer (pH5.0) formula (250ml):

[0040]

[0041] 2×Tm hybridization buffer

[0042]

[0043] Store at 4°C after filtration.

[0044] ExoSAP-IT kit was purchased from US USB Company.

[0045] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0046] 1. Sample DNA extraction:

[0047] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0048] 2. PCR amplification of samples to be tested

[0049] Design 2 pairs of primers and multiplex PCR to amplify 2 target sequences containing three common genotypes C451T, C478T, and G565T of the MC1R gene in one step, and the product sizes are 363bp and 228bp respectively. Table 3 shows.

[0050] First prepare the multiplex PCR pri...

Embodiment 3

[0091] The liquid phase chip of embodiment 3 different ASPE primers detects the SNP site of MC1R gene

[0092] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0093] Taking MC1R gene C451T, C478T and G565T 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 C451T, C478T and G565T, and the Tag sequence of the 5' end of the ASPE primer was It is selected from SEQ ID NO.1-SEQ ID NO.6, and correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.13-SEQ ID NO.18. 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.

[0094] Table 7 Design of liquid phase chip prepa...

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Abstract

The invention discloses a liquid phase chip and a specific primer for detecting MC1R gene mutation. The liquid phase chip mainly comprises: ASPE primers, microspheres coated by different anti-tag sequences, and an amplification primer, wherein each ASPE primer is composed of a tag sequence at a 5' end and specific primer sequences at a 3' end and aiming at target gene mutation sites, and the specific primer sequences are as follows: SEQ ID NO.7 and SEQ ID NO.8 aiming at a C451T site, SEQ ID NO.9 and SEQ ID NO.10 aiming at an C478T site, and / or SEQ ID NO.11 and SEQ ID NO.12 aiming at a G565T site. The coincidence rate of the detection result of the detection liquid phase chip disclosed by the invention with that of a sequencing method reaches as high as 100%, and parallel detection of a wild type and a mutant of a plurality of mutation sites is achieved.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a specific primer and a liquid phase chip for detecting M1R gene mutation. Background technique [0002] The melanocortin1 receptor (MC1R) is located on the long arm of chromosome 16 16q24.3, specifically between 89984286 and 89987384 base pairs on chromosome 16. MC1R is the smallest one (297-317 amino acids) in the melanocortin receptor (MCR) family, which are all G protein-coupled receptors with 7 transmembrane functional domains, and its natural ligand is black corticosteroid hormone peptide. It is now clear that the melanocortin system (MC) is widely involved in the regulation of a variety of physiological pathways, including pigmentation, feeding behavior, body weight and energy metabolism balance, anti-infection, sexual function and pain and other functions. MC1R is mainly expressed in melanocytes, and MC1R gene variation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B40/06C12Q1/68C12N15/11
Inventor 吴诗扬陈昌华
Owner SUREXAM BIO TECH
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