MLH1 Methylation Detection Kit

Product Name/Description	No. of Reactions*	Product Code
MLH1 Methylation Detection Kit	24	MLH1-RT24

* Includes all controls

About MLH1 Methylation

Lynch Syndrome (LS) is an autosomal dominant genetic syndrome that is associated with early-onset colorectal cancer (CRC), as well as other cancers. It is caused by germline mutations and inactivation of mismatch repair genes (MMR). The most common mutations are in MLH1 and MSH2. Most of these tumors are characterized by microsatellite instability (MSI; >90% of the LS tumors) and lack BRAF mutations. Yet, silencing of mismatch repair genes can also result from somatic promoter methylation in non-LS tumors.

Deng et al., analyzed the MLH1 promoter and defined two clinically-relevant regions (regions C and D), in which increased methylation correlates with gene silencing and MSI [1]. These results were further confirmed and extended, supporting MLH1 methylation analysis as a cost efficient pre-screening tool prior to genetic analysis for LS [2-5].

Figure 1: Schematic description of MLH1 promoter regions.

 

 

Kit Features

The MLH1 Promoter Methylation Detection Kit is a single-tube Methylation Sensitive Restriction Enzyme (MSRE)-based qPCR assay. Each reaction contains specific primers and probes for the amplification of regions C or D in MLH1 promoter (labeled by FAM), and a B2M internal control gene (labeled by CFO560/VIC).

Each DNA sample is tested in two (paired) reactions. DNA sample in one reaction tube is treated with methylation-sensitive restriction enzymes (RE) prior to the qPCR reaction, while in the second reaction DNA is amplified without the addition of restriction enzymes. The latter is used as reference for methylation ratio calculations. Restriction enzymes cut only the non-methylated sequences, making them unavailable for amplification. Differences in qPCR fluorescence signals of target MLH1 promoter regions C or D between the RE and the reference reaction are indicative of the levels of methylation in those regions. In addition, amplification of an internal input control (IC) gene is used to ensure that the same amount of sample is loaded. Using this approach, extracted DNA can be tested directly without the need for pre-treatment, such as bisulfite conversion.

Testing Procedure and Analysis

The testing procedure involves the following simple steps:

1. Isolation of DNA or RNA from tumor biopsies, paraffin-embedded sections (FFPE), or fresh frozen tumors.
2. Amplification using the provided reagents in the kit.
3. Data analysis and interpretation using real-time PCR software.

 Equipment & Materials

The MLH1 Methylation Detection Kit requires a real-time PCR instrument capable of detecting FAM and VIC fluorescent probes. The tests include reagents for PCR amplification/detection, as well as validated reaction controls. Columns and reagents for DNA isolation are not included.