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Kit, method and primers for analyzing methylation status of MLH1 promoter in DNA sample

A promoter and methylation technology, applied in recombinant DNA technology, DNA / RNA fragments, etc., can solve the problems of non-specific detection of methylated MLH1DNA primers and probes

Inactive Publication Date: 2015-11-04
常州杰傲病理诊断技术有限公司
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  • Abstract
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Problems solved by technology

However, currently available methods are non-quantitative, or they use primers and probes that do not specifically detect methylated MLH1 DNA, or that do not selectively target genomic regions of the promoter critical for MLH1 expression

Method used

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  • Kit, method and primers for analyzing methylation status of MLH1 promoter in DNA sample
  • Kit, method and primers for analyzing methylation status of MLH1 promoter in DNA sample
  • Kit, method and primers for analyzing methylation status of MLH1 promoter in DNA sample

Examples

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example 1

[0055] Example 1 Primers and Probes

[0056] Various embodiments are disclosed that allow the identification of reliable MLH1 methylation markers for improved sensitive diagnosis and prediction, diagnosis and staging of neoplastic diseases including CRC. To develop a reliable assay for the precise detection of MLH1 DNA methylation, a novel quantitative real-time system with primers and probes was designed for the exclusive amplification of methylated MLH1 DNA. These primers and probes specifically target regions of the MLH1 promoter region critical for its expression, as identified by G. Deng et al. (1999). As discussed in detail below, this assay has been found to provide an accurate determination of MLH1 methylation status in CRC tissue.

[0057] Although several methods for MLH1 DNA methylation were developed, these methods were mainly used for research purposes and none of these methods were successfully developed into accepted standard tests for clinical molecular diagno...

example 2

[0063] Example 2 Test for Detecting MLH1 Methylation

[0064] The assay for detecting MLH1 DNA methylation combines reference MLH1 methylation and ACTB normalization controls in a novel one-tube system. This design minimizes amplification bias between MLH1 and ACTB controls due to pipetting and variations in amplification efficiency in different PCR reactions. This assay includes primers and probes for MLH1 methylation and a 6-FAM / TAMRA probe selected from the primers and probes set forth in SEQ ID NO.: 1 to NO.: 9, and primers and probes for ACTB control. Needle. Probes for detection of MLH1 methylation and ACTB control were labeled with different reporter dyes, for example, 6-FAM and HEX, respectively. However, any suitable reporter now known or hereafter developed is within the scope of the present invention. Exemplary primers and VIC / TAMRA labeled probes for ACTB control are commercially available from Applied Biosystems (ABI) and Life Technologies.

[0065] Sample gen...

example 4

[0070] Example 4 Biological DNA sample analysis

[0071] The assay was tested to enable selective detection of methylated DNA in patient tissue and to establish clear and reliable boundaries for detection of MLH1 methylation. MLH1 methylation in 41 CRC tumors was analyzed by using the assay described in Example 2, and compared with the prior art method of Bettstetter (2007) ( figure 1 )Compare. CRC tumors were divided into two groups. Group 1 was negative for BRAF mutations and therefore negative for MLH1 DNA methylation. Group 2 was negative for MSI-H, MLH1 protein, and positive for BRAF mutation and should therefore undergo somatic MLH1 promoter hypermethylation.

[0072] Such as Figure 3A As shown, by using Bettstetter et al. (2007) ( figure 1 ) method for the analysis of methylated MLH1 DNA on CRC tumors. Percent methylation was calculated as published by Bettstetter et al. (2007). High levels of MLH1 methylation were detected in group 2 tumors. However, prior art...

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Abstract

The present invention belongs to the field of biotechnology, and in particular relates to a kit, a method and primers for analyzing methylation status of an MLH1 promoter in a DNA sample. The invention provides a kit for analyzing the methylation status of a neoplastic disease associated MLH1 promoter in a DNA sample. The kit comprises oligonucleotide primers, the oligonucleotide primers are complementary with at least part of the sequence of the MLH1 promoter in a zone from -248bp to -178bp relative to a transcription start site and overlap with the methylation sites in the zone. The present invention discloses accurate and sensitive test, composition and method for detection of differential methylation of genomic MLH1 promoter DNA in clinical sample. The test, composition and method can be used in allow diagnosis and symptom method; and the applicable characteristic is that the presenting level of methylation genomic MLH1 promoter DNA in the absence of a specific disorder is distinguished from illness of methylation genomic MLH1 promoter DNA level.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit, method and primers for analyzing the methylation status of MLH1 promoter in DNA samples. Background technique [0002] Lynch syndrome is an inherited form of colorectal cancer (CRC) associated with 2-5% of newly diagnosed CRC patients. Lynch syndrome is caused by germline mutations in DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2), causing high microsatellite instability and loss of MMR protein expression. However, only 15% of MSI-H CRCs are associated with Lynch syndrome and the remaining 85% are mostly sporadic in origin whereby mismatch repair deficiency is silenced by promoter hypermethylation of the MLH1 gene caused, and this is often combined with mutations in the gene BRAF. Thus, MLH1 promoter DNA methylation in combination with BRAF V600E mutations has been considered a reliable and standard-of-the-art molecular test to differentiate Lynch syndr...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 卢正斌李万帅
Owner 常州杰傲病理诊断技术有限公司
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