Analysis method for single nucleotide polymorphism

A detection method and specific technology, applied in the field of in vitro diagnostic nucleic acid analysis and detection, can solve problems such as unknown mutations, affecting analysis results, differences between melting curve peaks and actual sample amplification results, etc.

Inactive Publication Date: 2014-08-27
SHANGHAI TELLGEN LIFE SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The corresponding analysis of the high-resolution melting curve is an image analysis. All analyzes are based on the results of the melting curves of wild-type, mutant, and heterozygous reference products. During the current experiment, most of the mutant types are plasmids, and the amplification efficiency of plasmids and specimens There are still differences, and the final melting curve peak is different from the actual sample amplification results, which will lead to misjudgment of experimental results
In addition, HRM has very strict requirements for sample quantification. If the sample quantification is not accurate, the amplification efficiency of different samples will be different, which will affect the subsequent analysis results. The two homozygous states may be misjudged or interpreted as unknown. mutation
In the case of relatively accurate specimen quantification, it is also possible that the factors that inhibit PCR amplification in different specimens are different, resulting in different amplification efficiencies, which will also interfere with the final analysis results

Method used

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  • Analysis method for single nucleotide polymorphism
  • Analysis method for single nucleotide polymorphism
  • Analysis method for single nucleotide polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1, specific primer screening experiment

[0103] The purpose of the experiment: to introduce mutations, adjust the positions and bases of SNP sites and introduced mutation sites, and screen specific primers.

[0104] 1. Experimental materials

[0105] Primers were screened using wild-type, mutant and heterozygous samples to analyze primer sensitivity and specificity.

[0106] 2. Experimental grouping

[0107] Group 1: primer set TPPF_WT_1 (specific primer, referred to as "special") and TP-2-R2 (common primer, referred to as "general");

[0108] Group 2: primer set TPPF_WT_2 (special) and TP-2-R2 (general);

[0109] Group three: primer set TP-2_W_F3 (special) and TP-2-R2 (general);

[0110] Group 4: primer set TP-2_W_F4 (special) and TP-2-R2 (general).

[0111] 3. Primer sequences and related information of PCR products

[0112] TPPF_WT_1: 5'-TGTATGATTTTATGCAGG TT C-3' (SEQ ID NO: 3);

[0113] TPPF_WT_2: 5'-TGTATGATTTTATGCAGG TT C-3' (SEQ ID NO:...

Embodiment 2

[0129] Example 2, common primers and multiple allele-specific primers PCR amplification melting curve analysis

[0130] Experimental purpose: To investigate the results of melting curve analysis of common primers and multiple allele-specific primers in PCR amplification.

[0131] 1. Experimental materials

[0132] The wild-type, mutant and heterozygous samples were used to investigate the melting curve analysis results of different primer sets.

[0133] 2. Experimental grouping

[0134] Common primer set: group 1: TPMT2-PF01 (common) and TPMT2-PR01 (common), group 2: TPMT2-PF02 (common) and TPMT2-PR02 (common);

[0135] Multiple allele-specific primer set: Group 3: TP-2_W_F3 (special) and TP-2-R2 (general), TP-2-F (general) and Set2-M R2 (special) (the ratio of primers has been adjusted) .

[0136] 3. Primer sequences and related information of PCR products

[0137] TPMT2-PF01: 5'-AAATGTATGATTTTATGCAGGTTT-3' (Tm=55.6°C) (SEQ ID NO:8);

[0138] TPMT2-PR01: 5'-CACACCAACTAC...

Embodiment 3

[0163] Example 3, Peripheral Primer Amplification, Effect on Multiple Allele Specific Primer PCR Amplification Melting Curve Analysis

[0164] The purpose of the experiment: To investigate the effect of peripheral primer amplification on the melting curve analysis of PCR amplification with multiple allele-specific primers.

[0165] 1. Experimental materials

[0166] Use wild-type, mutant and heterozygous samples to investigate the melting curve analysis results of different primer sets;

[0167] 2. Experimental grouping

[0168] Group 1: TP-2_W_F3 (special) (1ul), Set2-M R2 (special) (1ul) and TP-2-F (common) (0.1ul), TP-2-R2 (0.1ul) (common) ( Peripheral primers cannot effectively amplify); the concentration of common primers is 10 times lower than that of specific primers;

[0169] Group 2: TP-2_W_F3 (0.5ul) (special), Set2-M R2 (0.5ul) (special) and TP-2-F (common) (0.5ul), TP-2-R2 (0.5ul) (common ) (peripheral primers can effectively amplify).

[0170] 3. Information ...

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Abstract

The invention relates to an analysis method for single nucleotide polymorphism (SNP), and discloses a novel method for determining known SNP. According to the method, specific primers are designed to amplify toward two different directions for to-be-detected gene templates; amplification efficiency of other two peripheral primers matching the specific primers is controlled, so that the whole PCR reaction system has only one specific PCR product in detection of wide type or homozygous mutant type samples and has two PCR products in detection of heterozygous samples and wild type, hybrid type and mutant type can be distinguished clearly. An SNP analysis method with stable results, good repeatability and wide applicable types is provided.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnostic nucleic acid analysis and detection; more specifically, the invention relates to a single nucleotide polymorphism analysis method. Background technique [0002] Single nucleotide polymorphism (Single nucleotide polymorphism, SNP) refers to changes such as conversion, transversion, insertion or deletion at a specific nucleotide position in genomic DNA. SNPs exist widely in the human genome, with approximately one SNP per 1000 bases. SNPs are the most common type of variation in human genetics. The occurrence of most diseases is related to the combined effect of environmental factors and genetic factors. It is generally believed that the disease is caused by the action of harmful environmental factors on the basis of the individual's genetic susceptibility. Different groups and individuals have differences in susceptibility, resistance to disease and other biological traits (such as re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2535/125
Inventor 郭安亮姚见儿王方金刘榴
Owner SHANGHAI TELLGEN LIFE SCI CO LTD
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