Nucleic acid sequence for detecting obesity risk site, kit and detection method thereof

A nucleic acid sequence and nucleotide sequence technology, which is applied in the field of nucleic acid sequence detection of obesity risk sites, can solve the problems of inability to meet the requirements of rapid clinical evaluation, inappropriate mutation site detection, and restricted development and application, so as to avoid Non-specific amplification problem, high accuracy, good detection effect

Inactive Publication Date: 2018-08-03
PRIMBIO GENES BIOTECH WUHAN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Sanger sequencing method is the gold standard for mutation analysis, which can find known and unknown mutation sites, but the instrument is expensive, the sensitivity is low, the operation is complicated, the cycle is long, and it is easy to contaminate; the ordinary PCR-RFLP method is simple in technology, cheap in price, and suitable for a small amount of samples laboratory testing, but it can only detect mutations with enzyme cleavage sites, and cannot be detected without enzyme cleavage sites, and there is a risk of false positives caused by PCR product contamination; gene chip technology has the characteristics of high throughput, miniaturization, automation, etc. , suitable for genome-wide mutation scanning, not suitable for mutation site detection of a single gene, low precision, and expensive, these shortcomings limit the development and application of these three methods in actual clinical work, and cannot meet the requirements of rapid clinical evaluation

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  • Nucleic acid sequence for detecting obesity risk site, kit and detection method thereof
  • Nucleic acid sequence for detecting obesity risk site, kit and detection method thereof
  • Nucleic acid sequence for detecting obesity risk site, kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Design of 5 obesity polymorphic site primers and verification templates

[0044] The ARMS primers selected in the present invention are designed for five obesity polymorphic sites (rs9939609, rs1558902, rs571312, rs17782313 and rs9356744). The selected ARMS primers are a set of primer combinations artificially added with mismatched mutation sites at the 3' end of a sequence and its vicinity. By screening primers capable of distinguishing alleles at a single site, the accuracy of PCR amplification is ensured. Highly specific.

[0045] After experimental verification, the specific detection primers for five obesity polymorphic sites were finally determined, and the nucleotide sequences were as follows:

[0046] FTO gene rs9939609 site wild-type upstream primer (PG1-F1), mutant upstream primer (PG1-F2) and common downstream primer (PG1-R1):

[0047] PG1-F1 (SEQ ID NO: 1): 5`-cttgcgactgctgtgaatgta-3`

[0048] PG1-F2 (SEQ ID NO:2): 5`-cttgcgactgctgtgaattct-3` ...

Embodiment 2

[0092] Example 2: Preparation of a kit for detecting 5 obesity polymorphic sites

[0093] For the five polymorphic sites, the wild-type upstream primers, mutant upstream primers and common downstream primers designed in Example 1 were used for synthesis, and SYBR Green I dye was used for staining, which is a dye that binds to dsDNA The dye with green excitation wavelength in the minor groove region of the double helix, in the free state, SYBR Green I emits weak fluorescence, but once combined with double-stranded DNA, the fluorescence is greatly enhanced, so the fluorescence intensity of SYBR Green I is related to the amount of double-stranded DNA Correlation, so the amount of double-stranded DNA present in the PCR system can be detected based on the fluorescent signal.

[0094] A kit for detecting obesity risk sites, including 10 PCR reaction solutions, positive controls, negative controls, and chromogenic reagents.

[0095] There are 10 kinds of PCR reaction liquids among t...

Embodiment 3

[0105] Embodiment 3: Detect 5 obesity polymorphic sites with the kit prepared by the present invention

[0106] For the determination of genomic DNA, almost any biological sample that contains genomic DNA (eg, impure red blood cells) can be used. For example, and without limitation, genomic DNA may be conveniently obtained from blood, semen, saliva, tears, urine, feces, sweat, buccal cells, skin or hair. In order to utilize DNA assays, the target nucleic acid must be obtained from a cell or tissue expressing the target sequence.

[0107]The sample to be tested in this embodiment is DNA extracted from 10 cases of oral swabs collected by Wuhan Liangpei Gene Biotechnology Co., Ltd., and its quantification is used as a template for PCR detection. For the main operation steps of DNA extraction, refer to Tiange Company’s buccal swab DNA extraction kit to extract genomic DNA from samples. The purity and concentration of the extracted genomic DNA were calculated by an ultraviolet sp...

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Abstract

The invention discloses a nucleic acid sequence for detecting an obesity risk site, a kit and a detection method thereof. A nucleic acid sequence combination disclosed by the invention is proposed specific to five polymorphic sites (rs9939609, rs1558902, rs571312, rs17782313 and rs9356744), and comprises a wild upstream primer, a mutant upstream primer and a universal downstream primer of each site. The kit for rapidly detecting the obesity risk site and prepared from the nucleic acid sequence combination has the characteristics of convenience in use, easiness and convenience in operation, high automation degree, lower pollution in an operation process, good detection effect, high sensitivity, high specificity, high accuracy and high precision. The detection method disclosed by the invention is convenient and rapid to use through adoption of a complete closed-tube operation; a detection result is obtained by directly exploring a fluorescent signal value in a PCR (Polymerase Chain Reaction) process, so that PCR posttreatment or electrophoresis detection is not required, the technical problems of great probability of pollution and false positive value generation in the conventional PCR technology are solved, and non-specific amplification can be effectively avoided; the detection method is suitable for large-scale sample detection.

Description

technical field [0001] The invention relates to the fields of medicine and biotechnology, in particular to a nucleic acid sequence for detecting obesity risk sites, a kit and a detection method thereof. Background technique [0002] Obesity is a chronic disease closely related to genetic factors and lifestyle. Different individuals have different high-risk factors for obesity due to their different genetic constitutions and living environments, and there are also differences in effective forms of weight control. Therefore, understanding one's own genetic status can not only prevent obesity and overweight through dietary structure and lifestyle adjustments. [0003] The body fat mass and obesity-related gene (FTO) is located on chromosome 16q12, encoding a demethylase. Studies have shown that different genotypes of FTO are closely related to the incidence of obesity. People with FTO rs9939609AT and AA genotypes People with the rs9939609TT genotype have a significantly higher...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2563/107
Inventor周艳琳
OwnerPRIMBIO GENES BIOTECH WUHAN CO LTD