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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  • Description
  • Claims
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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 cont

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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

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Example Embodiment

[0044] Example 1

[0045] The primers for detecting 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 NO 2 is labeled with biotin, and the sequencing primer SEQ NO 3, the base sequence of which 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 to T, which will cause 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 on the Therefore, the amplification primers and sequencing primers used in the PCR of the present invention are all self-designed, fully considering the characteristics of the DNA after sulfite treatment, and the bindi...

Example Embodiment

[0060] Example 2: MGMT promoter methylation detection method

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

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

[0063] (2) The template DNA is subjected to sulfite treatment, and purified and recovered;

[0064] (3) Using the purified and recovered product in step (2) as a template, perform 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-stranded purification, it is sequenced in a pyrophosphate sequencer;

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

[0067] 2. Step (1) DNA extraction: you can select extraction reagents well-known to those skilled in the industry for DNA extraction, or use kits developed by biotechnology companies for DNA extraction.

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

[0069] ...

Example Embodiment

[0079] Example 3: Clinical sample detection

[0080] Select 36 clinical tumor samples to be tested, and use the kit of the present invention for testing. Sample DNA extraction, sulfite 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 analysis.

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

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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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