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Reagent and method for detecting MGMT gene promoter methylation

A technology for detection of reagents and promoters, applied in the fields of life science and biology, can solve problems such as variation of CG content and TM value, influence on acquisition, etc., and achieve high detection rate, high accuracy, and high throughput

Inactive Publication Date: 2015-06-24
FUZHOU ADICON CLINICAL LAB INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] After the DNA sequence is treated with sulfite, part of the C bases will be converted into T, resulting in a large degree of variation in the CG content and TM value in the sequence region, which will affect the conventional primer design software to obtain ideal results on the sequence. Primer sequence

Method used

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  • Reagent and method for detecting MGMT gene promoter methylation
  • Reagent and method for detecting MGMT gene promoter methylation
  • Reagent and method for detecting MGMT gene promoter methylation

Examples

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Embodiment 1

[0045] The primers for detecting the methylation of the MGMT promoter include a pair of specific amplification primers SEQ NO 1 and SEQ NO 2, wherein the 5-end of SEQ NO2 is labeled with biotin, and the sequencing primer SEQ NO 3, its base sequence is as follows:

[0046] SEQ NO 1: 5'-GYGTTTYGGATATGTTGG-3';

[0047] SEQ NO 2: 5'-CRAAACRACCCAAACACTCAC-3';

[0048] SEQ NO 3: 5'-GATAGTTYGYGTTTTTAGAA-3';

[0049] When designing primers, it is considered that after the DNA sequence is treated with sulfite, some of its C bases will be converted into T, resulting in a large degree of variation in the CG content and TM value in the sequence region, which will affect the conventional primer design software in its sequence. Get the ideal primer sequence. Therefore, the amplification primers and sequencing primers used in PCR in the present invention are all self-designed, fully considering the characteristics of the DNA after sulfite treatment, and the binding site of the 3-terminal o...

Embodiment 2

[0060] Example 2: MGMT promoter methylation detection method

[0061] 1. The MGMT promoter methylation detection method is as follows:

[0062] (1) extract the DNA in the sample;

[0063] (2) subjecting the template DNA to sulfite treatment, and purifying and recovering;

[0064] (3) Using the purified recovered product in step (2) as a template, carry out two rounds of PCR amplification according to the specific amplification primers (SEQ NO1, SEQ NO2);

[0065] (4) After the PCR product is subjected to single-strand purification, it is placed in a pyrosequencer for sequencing;

[0066] (5) Obtain the methylation status of the MGMT promoter of the sample according to relevant software.

[0067] 2. Step (1) DNA extraction: You can choose extraction reagents well-known to those skilled in the art for DNA extraction, or use a kit developed by a biotechnology company for DNA extraction.

[0068] 3. Step (2) sulfite treatment and purification recovery methods are as follows: ...

Embodiment 3

[0079] Embodiment 3: clinical sample detection

[0080] Select 36 cases of clinical tumor samples to be tested, and use the kit of the present invention for detection. Sample DNA extraction, sulfurous acid treatment, purification and recovery, PCR amplification, pyrosequencing and result analysis were carried out according to the method in Example 2. The results of the methylation status of the MGMT gene promoter of 36 samples are shown in Table 1:

[0081] Table 1

[0082]

[0083] The value in the column of methylation percentage of each sample in Table 1 represents the average methylation percentage value of the 5 detection sites on the MGMT promoter, which is given by the software PyroMark CpG after analysis.

[0084] It can be seen from Table 1 that the methylation status of the MGMT gene promoter in the above 36 tumor samples can be successfully and accurately detected using the detection method of PCR combined with pyrosequencing of the present invention. Therefore...

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Abstract

The invention discloses a reagent and a method for detecting MGMT gene promoter methylation. The reagent comprises (1) specific amplimers as shown in SEQ NO 1, SEQ NO2 of which the 5th ends are marked with biotin, as well as specific sequencing primer SEQ NO3; and (2) a PCR amplified reaction reagent, a sulfite treatment reagent and a pyrophosphoric acid sequencing related reagent. The reagent and the method disclosed by the invention have the advantages of short detection cycle, high specificity, high accuracy, low pollution risk and the like, and can be used for judging the MGMT gene promoter methylation state of a to-be-detected sample, and the detection result is beneficial to prediction of early-phase related tumor diseases, and can be used for guiding clinical doctors to use alkylating agent medicines according to individual differences of patients so as to realize individual treatment and for guiding doctors to carry out prognosis treatment of related diseases.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and relates to a reagent and method for detecting methylation of MGMT gene promoter. Background technique [0002] Improving the efficacy of conventional chemotherapy drugs by modulating DNA repair mechanisms is an important area of ​​cancer research. o 6 -Methylguanine-DNA methyltransferase (MGMT) repairs O 6 - The special mechanism of action and the characteristics of suicide enzymes in the damage of alkylguanine make it of great significance in the early diagnosis of tumors and the prediction of tumor sensitivity to alkylating agents. [0003] MGMT, as a DNA repair protein, can remove guanine O from DNA 6 The mutagenic and cytotoxic alkylated adducts at the site can restore the damaged guanine, thereby protecting cells against the damage of the alkylated group, and the expression of the MGMT gene regulated by promoter methylation Silencing reveals an important mutation pathway...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q1/6869C12Q2600/106C12Q2600/154C12Q2523/125C12Q2535/122C12Q2565/301
Inventor 陈奕磊林筱剑王淑一
Owner FUZHOU ADICON CLINICAL LAB INC
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