Reagent and method for detecting MGMT gene promoter methylation

[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 binding site of the 3 end of the sequencing primer is directly located at the first site to be tested The previous base.

[0050] The MGMT promoter methylation detection kit includes: 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, whose base sequence is as follows Show:

[0051] SEQ NO 1: 5'-GYGTTTYGGATATGTTGG-3';

[0052] SEQ NO 2: 5'-CRAAACRACCCAAACACTCAC-3';

[0053] SEQ NO 3: 5’-GATAGTTYGYGTTTTTAGAA-3’;

[0054] The MGMT gene promoter methylation detection kit also includes the following reagents:

[0055] (1) PCR amplification reagents: 10*PCR Buffer, 2mM dNTP, 5*Q Solution Buffer, 5U/ul Taq enzyme, 10uM amplification primers (SEQ NO1, SEQ NO 2) and sterilized water;

[0056] (2) Sulfite treatment reagents: Bisulfite Mix, RNase free water, DNA Protect Buffer;

[0057] (3) Single-stranded purification reagents: streptavidin beads, 70% (V/V) ethanol, 8mg/ml NaOH solution, 1×Wash Buffer, binding buffer, annealing buffer;

[0058] (4) Sequencing reagents: DNA polymerase, ATP sulfurylase, luciferase, ATPase, substrate APS, luciferin and dNTPs and sequencing primers (SEQ NO3);

[0059] (5) Positive control: contains MGMT promoter DNA after sulfite treatment; negative control: does not contain MGMT promoter DNA after sulfite treatment

[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] The sulfite treatment reaction system of a sample is 140ul: 10ul DNA, 10ul RNase free water, 85ul Bisulfite Mix, 35ul DNA Protect Buffer.

[0070] The sulfite treatment reaction procedure is: 95°C 5min, 60°C 25min, 95°C 5min, 60°C 85min, 95°C 5min, 60°C 175min.

[0071] The method of purification and recovery of a sample is as follows: add 310ul Buffer BL (including 10ug/ml carrier RNA) to the above sulfite reaction mixture, shake and mix; add 250ul absolute ethanol, shake for 15s, centrifuge and mix at low speed; Transfer the above mixture to EpiTect spin columns, centrifuge at full speed for 1 min, discard the waste; add 500ul Buffer BW, centrifuge at full speed for 1 min, discard the waste; add 500ul Buffer BD, leave it at room temperature for 15 minutes, centrifuge at full speed for 1 min, discard the waste; add 500ul Buffer Centrifuge at full speed for 1 min and discard waste liquid; centrifuge at full speed for 1 min and discard waste liquid; open the lid of the spin column and place it in a metal bath at 56°C for 5 min; place the spin column on a clean 1.5 mL centrifuge tube and transfer it to the membrane Add 20μL Buffer EB dropwise to the center at 12000rpm for 1min, and collect the resulting liquid for the next step of PCR or store at -20°C.

[0072] 4. The PCR amplification method described in step (3) is as follows:

[0073] The first round of PCR system for a sample is 25ul: 10*PCR Buffer 2.5ul, 2mM dNTP 2.5ul, 5*Q Solution Buffer 5ul, 10uM primer SEQ NO10.5ul, 10uM SEQ NO20.7ul, 5U/ul Taq enzyme 0.125ul , 11.675ul sterile water, 2ul template.

[0074] The first round of PCR reaction program: 94 ℃ 30s, 48 ​​℃ 30 s, 72 ℃ 1 min, 49 cycles, 72 ℃ 10 min.

[0075] The second round of PCR system for one sample is 50ul, in which all PCR reagent components are doubled; the number of second round amplification cycles is set to 38, and the remaining reaction conditions remain unchanged.

[0076] Since the sample DNA will be degraded to a certain extent during the sulfite treatment process, which will affect the later detection, the present invention adopts two rounds of PCR. This design greatly improves the detection rate.

[0077] 5. The specific steps described in step (4) are: mix the 50ul PCR product obtained with 3ul streptavidin bead and 47ul binding buffer, incubate at room temperature for 10-15 minutes, and shake 2 to 3 times during this period to prevent the bead from sinking . Then use the Vaccuum prep tool in the Vacuum prep workstation to absorb the bead, and put the Vaccuum prep tool with the bead into 70% (V/V) ethanol, Denaturation Solution, 1×Wash Buffer for about 10 seconds, and then the Vaccuum prep tool Put into a 96-well plate containing 49ul annealing buffer and 1ul sequencing primer (SEQ NO3), release the bead, place the 96-well plate at 80°C for 2 minutes, and then cool to room temperature to obtain the single-stranded purification required for the sequencing reaction product.

[0078] 6. The result analysis described in step (5) is specifically: taking the sulphite-treated MGMT promoter sequence as the standard sequence and inputting it into the methylation detection software PyroMark CpG, and the MGMT of the sample is obtained through the software analysis Promoter methylation status.

[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 invention can not only realize the qualitative and quantitative determination of site methylation at the same time, but also ensure a higher PCR amplification success rate through optimized primer amount ratio and two rounds of PCR. In addition, due to the combined use of pyrosequencing technology, this detection method has the advantages of high accuracy, high throughput, short detection cycle, and low pollution risk. The final analysis results can help clinicians better develop related diseases. treatment.