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Primers, kit and method to detect methylation of MLH1 promoter gene based on pyrosequencing

A pyrosequencing method and pyrosequencing technology, applied in the fields of life sciences and biology, can solve the problems of high requirements, cumbersome operation, and high probe cost, and achieve the effects of high accuracy, high throughput, and high detection rate

Inactive Publication Date: 2019-09-20
北京艾迪康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

Among them, the MSP method uses PCR amplification detection to determine whether there is methylation in the sample. This method is practical and common, but it cannot achieve quantitative detection, and there is a high risk of false positives; the BSP method mainly uses PCR combined with sanger sequencing technology. However, due to its complicated operation, it is not suitable for large-scale detection, and the number of clones selected at the same time may affect the detection results, so BSP can only be regarded as a semi-quantitative method; MS-HRM is a single-base sequence The difference of the melting curve is transformed into the difference of the melting curve, so as to judge whether there is methylation. This method has high requirements on the instrument, and needs a fluorescent quantitative PCR instrument with an HRM module. At the same time, this method can only analyze the overall methylation status of the detection fragment. , but cannot clarify the methylation status of each CpG site; the fluorescence quantitative method is based on the technology developed by MSP, mainly adding TaqMan probes in the detection, thus ensuring high sensitivity and accuracy, but it is for The detection of multiple methylation sites can only be analyzed as a whole, and the probe cost is high, so this method is more suitable for the detection of a large number of samples at a few sites

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  • Primers, kit and method to detect methylation of MLH1 promoter gene based on pyrosequencing
  • Primers, kit and method to detect methylation of MLH1 promoter gene based on pyrosequencing
  • Primers, kit and method to detect methylation of MLH1 promoter gene based on pyrosequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] MLH1 promoter methylation detection method, including: a pair of specific amplification primers SEQ NO1 and SEQ NO 2, wherein the 5' end of SEQ NO1 is labeled with biotin, and SEQ NO 2 is also used as a sequencing primer, and its base sequence is as follows Show:

[0042] SEQ NO1: 5'Bio-AAGGGTGGGGTTGGATGG-3';

[0043] SEQ NO 2: 5'-GCGATACGATTCACCACTATCTC-3';

[0044] Further, the concentration ratio of SEQ NO1:SEQ NO 2 is 0.5 μM:0.5 μM.

[0045] Specifically, the MLH1 gene promoter methylation detection method includes the following reagents:

[0046] (1) Sulfite treatment reagents: Bisulfite Mix, RNase free water, DNA ProtectBuffer;

[0047] (2) 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;

[0048] (3) Single-chain purification reagents: streptavidin beads, 70% (V / V) ethanol, 8mg / ml NaOH solution, 1×WashBuffer, binding buffer, annealing buffer;

[00...

Embodiment 2

[0051] Example 2: MLH1 promoter methylation detection method detection process

[0052] 1. The method of using the MLH1 promoter methylation detection method is as follows:

[0053] (1) extract the DNA in the sample;

[0054] (2) subjecting the template DNA to sulfite treatment, and purifying and recovering;

[0055] (3) using the purified recovered product in step (2) as a template, and carrying out two rounds of PCR amplification according to the specific amplification primers (SEQ NO1, SEQ NO 2);

[0056] (4) After the PCR product is subjected to single-strand purification, it is placed in a pyrosequencer for sequencing;

[0057] (5) Obtain the methylation status of the MLH1 promoter of the sample according to relevant software.

[0058] 2. Step (1) DNA extraction: You can choose extraction reagents well-known to those skilled in the art for DNA extraction, or use a method developed by a biotechnology company for DNA extraction.

[0059] 3. Step (2) sulfite treatment an...

Embodiment 3

[0068] Embodiment 3: clinical sample detection

[0069] Take 12 cases of clinical tissue samples to be tested, and use the method of the present invention to detect. 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 MLH1 gene promoter of the 12 samples are shown in Table 1:

[0070] Table 1 Tumor sample detection results

[0071]

[0072] Note: 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 MLH1 promoter, which is given by the software PyroMark CpG analysis.

[0073] figure 2 It is the result figure of agarose gel electrophoresis after PCR amplification of the present invention.

[0074] image 3 is the pyrosequencing result of tumor sample 1.

[0075] From the results of Example ...

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Abstract

The invention discloses primers, kit and method to detect methylation of MLH1 promoter gene based on pyrosequencing. The primers include a pair of specific amplification primers SEQ NO. 1 and SEQ NO. 2,wherein biotin is marked at 5' end of the SEQ NO. 1, and the SEQ NO. 2 also acts as a sequencing primer. The primers, kit and method can provide qualitative and quantitative determinations on locus methylation; compared with other conventional detection methods, the method herein has the advantages of high accuracy, high detection rate, high flux, short detection rate, low pollution risk and the like. The detection results are more helpful for clinicians to better carry out diagnosis, treatment and prognostic assessment on related diseases.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and relates to a method for detecting methylation of MLH1 gene promoter. Background technique [0002] DNA methylation (DNA methylation) is one of the important modifications of epigenetics. Its methylation generally occurs at CG-linked dinucleotide sites (CpGs), and changes chromatin structure, DNA structure and stability, etc. play an important role in the regulation of gene expression. With the development of epigenetics, people realized that tumors are not only genetic diseases, but also closely related to epigenetics such as abnormal gene regulation caused by abnormal DNA methylation. In normal cells, the CpG islands in the promoter region are unmethylated, while most of the scattered CpG island dinucleotides are mostly methylated. Tumors are often accompanied by a decrease in the overall methylation level of the genome and an abnormal increase in the methylation level of cert...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6869C12N15/11
CPCC12Q1/6886C12Q1/6869C12Q2600/154
Inventor 牛林梅王淑一
Owner 北京艾迪康医学检验实验室有限公司