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Method and reagent kit for detecting mutation of human TERT gene promoter

A technology of promoters and kits, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as reduced amplification efficiency, inaccurate results, and difficulty in fragment amplification

Inactive Publication Date: 2015-12-30
杭州吉辰君创医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages of the dye method: Since the combination of SYBRGreen and dsDNA is only structurally specific but not sequence specific, in addition to binding to specific target sequence amplification products, it can also dimerize with primers formed during PCR amplification Body, non-specific amplification product combination, resulting in the reduction of amplification efficiency and inaccurate results
In addition, the promoter region of the TERT gene is a non-coding region, and the GC content in the sequence is very high, so it is very difficult to amplify the fragment

Method used

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  • Method and reagent kit for detecting mutation of human TERT gene promoter
  • Method and reagent kit for detecting mutation of human TERT gene promoter
  • Method and reagent kit for detecting mutation of human TERT gene promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 kit detection steps

[0044] 1. Refer to the following PCR 96-well plate layout table:

[0045]

[0046]

[0047] Add 10μLC228T reaction mixture to each reaction space in columns 1-4 of the 96-well plate (the volume ratio of TERTC228T forward primer, TERTC228T reverse primer and 2×PCR master mix containing fluorescent dye in TERTC228T reaction mixture is 1.25: 1.25:7.5, the concentrations of TERTC228T forward primer and TERTC228T reverse primer in TERTC228T reaction mixture are both 4.8μM), 10μLC250T reaction mixture (TERTC250T forward primer, TERTC250T reverse The volume ratio of the primer and the 2×PCR master mix containing fluorescent dye in the TERTC250T reaction mixture is 1.25:1.25:7.5, and the concentrations of the TERTC250T forward primer and the TERTC250T reverse primer in the TERTC250T reaction mixture are both 4.8 μM), 14 μL of internal standard reaction mixture was added to each reaction space in columns 9-12 (every 14 μL of TERT inter...

Embodiment 2

[0054] Embodiment 2 result judgment and analysis method

[0055] 1. Determination of mutation results: first determine the mutation Ct value (abbreviated as A) of each C228T reaction system or C250T reaction system, and then determine the Ct value of the internal standard reaction system of the sample and the Ct value of the positive quality control product. The Ct value of the internal standard reaction system plays a vital role in the calculation of the mutation ratio in the later stage. For the specific role, please refer to the next step. The Ct value of positive quality control product 1 and positive quality control product 2 can directly determine the validity of the result. It belongs to the quality control point in the reaction system. Different Ct values ​​represent different degrees of mutation, and the detection results can be analyzed according to the result judgment criteria (Table 1):

[0056] Table 1 Results Judgment Criteria

[0057]

[0058] *Calculatio...

Embodiment 3

[0064] The optimization of embodiment 3 reaction system

[0065] 1. Confirmation of the amount of genomic DNA of the sample to be tested: add the 10ng / μl sample human genomic DNA in Example 1 to the R132H reaction system with 4 additions of 1ng, 5ng, 10ng, and 50ng respectively, and finally confirm that the concentration is 50ng (5μl). 10ng / / μl template) as the final addition amount of C228T reaction system and C250T reaction system, this addition amount can exclude the influence of human operation factors.

[0066] 2. Exploring the number of cycles: A represents the cycle number of the second stage in step 4 of embodiment 1; B represents the cycle number of the third stage in step 4 of embodiment 1, according to the following combinations, the final verification results show that A is 18 and B The collocation of 24 is the best response process.

[0067] A:18repeats(19total)B:24(23total)

[0068] A:16repeats(17total)B:26(26total)

[0069] A:14repeats(15total)B:28(29total)...

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Abstract

The invention discloses a method and a reagent kit for detecting mutation of a human TERT gene promoter. The reagent kit includes a TERT C228T reaction mixed solution and / or a TERT C250T reaction mixed solution, and further includes a TERT internal standard reaction mixed solution. The method comprises the following steps that firstly, human genome DNA which is extracted from a sample and serves as a template is added into the TERT C228T reaction mixed solution and / or the TERT C250T reaction mixed solution, the human genome DNA same with that in the system is added into an internal standard reaction system, and fluorogenic quantitative PCR amplification is conducted; secondly, Ct values are analyzed. According to the detecting method and the reagent kit, influences of adverse conditions that the GC content of a TERT gene promoter sequence is high and amplification is difficult are overcome, C228T locus mutation and C250T locus mutation are effectively identified, and the detecting sensitivity reaches 1%.

Description

technical field [0001] The invention relates to a method for detecting gene mutation and its kit, in particular to a method for detecting human TERT gene promoter mutation and its kit. Background technique [0002] Telomerase is a ribonucleoprotein complex composed of RNA and protein, belonging to reverse transcriptase, which is closely related to the regulation mechanism of telomeres and plays a key role in maintaining cell immortalization and carcinogenesis. Telomerase reverse transcriptase (telomerase reverse transcriptase, TERT) is an important catalytic part of telomerase. The TERT gene is located on human chromosome 5, and the two promoter mutation sites of the gene are 1,295,228C>T and 1,295,250C>T (referred to as C228T and C250T, respectively) are particularly common, and these two mutations have been shown to enhance transcriptional activity of the TERT promoter. Mutations in the TERT promoter region do not exist in normal human subjects and public gene datab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2545/113C12Q2563/107
Inventor 阎海焦雨辰王晓月王思振熊梓锴
Owner 杭州吉辰君创医学检验实验室有限公司
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