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Rs 3909184 detection genotyping kit based on AllGlo probe and genotyping method thereof

A kit and probe technology, applied in the field of single nucleotide polymorphism, can solve the problems of long cycle, insufficient specificity, cumbersome experimental operation, etc., achieve high specificity and sensitivity, low detection price, and promising application prospects broad effect

Inactive Publication Date: 2016-04-13
ZHONGSHAN HOSPITAL XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the direct sequencing method is currently the most intuitive and relatively accurate SNP typing method, but its steps are many and scattered, the cost is high, the workload is heavy, the cycle is long, and the price is expensive, so it is not suitable for large samples and multiple loci. Detection; Restriction Fragment Length Polymorphism (RFLP), as the earliest DNA labeling technology, has low requirements for instruments, and only needs a PCR instrument and an electrophoresis instrument to carry out the experiment, but its experimental operation is cumbersome and the detection cycle is long , not suitable for large sample detection; Tagman fluorescent probe method has good accuracy, but its design and synthesis procedures are complicated, and it is not suitable for typing of multiple loci and a small number of samples, especially loci with low frequencies; amplification block mutation The system (ARMS method) is fast and simple, but it cannot be operated in a closed tube, and it is difficult to achieve high-throughput and automation for gene polymorphism analysis; high-resolution melting curve analysis (HRM) has the advantages of simplicity and speed, but the method is specific Insufficient, few instrument choices

Method used

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  • Rs 3909184 detection genotyping kit based on AllGlo probe and genotyping method thereof
  • Rs 3909184 detection genotyping kit based on AllGlo probe and genotyping method thereof
  • Rs 3909184 detection genotyping kit based on AllGlo probe and genotyping method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: The AllGlo probe-based rs3909184 detection and typing kit of the present invention includes the following components: real-time fluorescent quantitative PCR reagent.

[0037] ①Reagents for real-time fluorescent quantitative PCR include the following components: 10×Taq buffer (10×Taq buffer includes 100mmol Tris-HCl and 500mmol KCl), 10mmol MgCl 2 , 5u / μL TaqHotstart DNA polymerase, 10mmoldNTPs mixture, 50×LowROX, 100mL nuclease-free water, 10μmol / Lrs3909184 specific forward primer, 10μmol / Lrs3909184 specific reverse primer and AllGlo probe.

[0038] ② Positive controls include: positive homozygous control substance 1, positive homozygous control substance 2, and positive heterozygous control substance. The positive homozygous control substance 1 is a DNA sample typed as GG by rs3909184, the positive homozygous control substance 2 is a DNA sample typed as CC by rs3909184, and the positive heterozygous control substance is a DNA sample typed as GC by rs3909184 ...

Embodiment 2

[0041] The detection and typing of rs3909184 in peripheral blood includes the following steps:

[0042] 1. Collect EDTA anticoagulant peripheral blood: Take 2 mL of fresh blood samples and put them in EDTA anticoagulant tubes and divide them into multiple tubes. Each tube is divided into 400-500 μL, take 200 μL for DNA extraction, and store the rest of the whole blood samples at -80°C Freeze.

[0043] 2. Extract DNA: Use the Genomic Blood DNA Extraction Kit (DP348) produced by Tiangen Biotechnology Co., Ltd. to extract DNA from the sample (whole blood) according to the operating instructions, and use 200 μL of LEDTA anticoagulated whole blood according to the instructions, and finally use 50 μL DNA was dissolved in the elution buffer TB, and the concentration and purity were measured on the nucleic acid spectrophotometer NanoDrop2000. The ratio of purity A260 / A280 was taken to be between 1.7 and 1.9, and the extracted DNA with a concentration of 10 to 50 ng / μL was used for SNP...

Embodiment 3

[0058] Example 3. SNP detection and typing of tissue or cell samples

[0059] 1. Sample handling:

[0060] a. Tissue: Animal tissue (the amount of spleen tissue should be less than 10mg) should be crushed and processed into a cell suspension, then centrifuged at 10000rpm (~11200×g) for 1min, the supernatant was poured out, and 200ul buffer GA (Tiangen DNA extraction reagent) was added. box DP304), shake until completely suspended;

[0061] b. Adherent cultured cells should be processed into cell suspension first, then centrifuged at 10,000 rpm (~11,200×g) for 1 min, drain the supernatant, add 200 μL of buffer GA (Tiangen DNA Extraction Kit DP304), shake until completely suspended ;

[0062] 2. Tissue, cell DNA extraction and enrichment:

[0063] Use the DNA extraction kit (DP304) produced by Tiangen Biotechnology Co., Ltd. to extract tissue or cell DNA according to the operating instructions, and finally dissolve the DNA with 50 μL of elution buffer TE, and measure the conc...

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Abstract

The invention relates to single nucleotide polymorphism, in particular to an rs 3909184 detection genotyping kit based on an AllGlo probe and a genotyping method thereof. The kit comprises a real-time fluorescence quantification PCR (polymerase chain reaction) reagent, positive control and negative control, and rs 3909184 is an SNP (single nucleotide polymorphism) locus serial number provided by NCBI(National Center of Biotechnology Information). The detection genotyping method includes the steps that DNA in an EDTA anticoagulant whole blood sample is extracted by adopting a conventional method; the real-time fluorescence quantification PCR reagent provided by the rs 3909184 detection genotyping kit based on the AllGlo probe is used for conducting real-time fluorescence quantification PCR amplification on DNA; the rs 3909184 SNP locus is genotyped according to detected fluorescence signals. On the premise of keeping high specificity and sensibility of the AllGlo probe, the detection price is lower than that of a direct sequencing method, the process is simpler, and consumed time is shorter.

Description

technical field [0001] The present invention relates to single nucleotide polymorphism (SNP), in particular to an AllGlo probe-based rs3909184 detection and typing kit and typing method thereof. Background technique [0002] Single nucleotide polymorphism (singlenucleotide polymorphism, SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. It is the most common type of heritable variation in humans. SNPs widely exist in the human genome, with an average of 1 in every 500-1000 base pairs, and it is estimated that the total number can reach 3 million or more. As a third-generation genetic marker, SNP is closely related to many phenotypic differences, drug susceptibility and disease susceptibility. The large number of SNPs in the genome makes it a powerful tool and plays a very important role in disease localization and cloning, drug design and testing, and basic biological research. [0003] Individualized di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q1/6858C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 张忠英方宜臻
Owner ZHONGSHAN HOSPITAL XIAMEN UNIV
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