CpGenome & CpG WIZ
Methlaytion-Specific PCR* (MSP) System

Tools for Measuring Levels of Silencing-associated CpG Methlyation in DNA at Specific Genes

CpGenome DNA Modification Kit

The CpGenome DNA Modification Kit is used to create methylation-specific sequence alterations in any DNA sample. Using a bisulfite treatment process, all unmethylated cytosines are converted to uracils; methylated cytosines remain unaltered. Thus, the methylation state of the DNA in vivo will determine the sequence of the DNA following bisulfite treatment. Methylation-specific PCR (MSP) is then employed to detect which sequence is now present by using primers specific to each possible sequence. These primers can be designed using CpG WARE or other software; alternatively, CpG WIZ kits are available that supply the necessary primers to detect the methylation state of specific targets.

CpGenome FAST DNA Modification Kit

CpGenome FAST DNA Modification Kit employs the same principles as the original CpGenome DNA Modification Kit, but incorporates a DNA spin column for ease of use and more rapid processing of DNA samples.

Detection of the Methylation State of the p16 Gene

Methylation Specific PCR (MSP) of the p16 gene in two invasive carcinomas, a squamous intraepithelial lesion (SIL), and an adenocarcinoma of the cervix. Each numbered set has paired MSP reactions specific for both the unmethylated (U) and methylated (M) alleles of the p16 CpG island. The presence or absence of the “M” MSP amplicon is indicative of the methylation state of the p16 gene in the sample. The results indicate that both invasive carcinomas and the SIL sample are heterozygous for methylation while the adenocarcinoma sample is clearly homozygous for the unmethylated state at the p16 locus. *This product is optimized for use with Polymerase Chain Reaction (PCR) process which is covered by patents owned by Roche Molecular Systems, Inc. No slicense or rights under these patents to use the PCR process is conveyed expressly or by implication to the purchaser of this product.

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How Methylation-specific PCR Works

Step 1. Bisulfite Treatment — The first step in MSP involves the chemical conversion of all unmethylated cytosines to uracil using the reagents in the CpGenome Universal DNA Modification Kit (cat. #S7820). Methylated cytosines remain unaltered in the process. Thus, the sequence of the DNA after bisulfite treatment will be different depending on the original methylation state of the DNA.

Step 2. PCR with Methylation Specific Primer Sets — Methylation specific PCR is used to determine which sequence is present after bisulfite treatment. Primers to the unmethylated and methylated sequences must be designed, such that mismatches are created depending on which sequence is present to prevent mispriming between the primer sets and the undesired target DNA. A typical experiment will involve performing 2 PCR reactions using the same bisulfite-treated template DNA. One reaction uses primers (“U” primer set) designed to anneal to the sequence present if the DNA is unmethylated, and the other reaction will include primers (“M” primer set) designed to anneal to the sequence if the DNA is methylated.

Step 3. Gel Analysis — The PCR products are run on an agarose gel stained to visualize the DNA. If the sample DNA was originally unmethylated prior to modification, only the “U” primer set will produce an amplification product. Conversely, a product will only be produced with the “M” primer set if the DNA was originally methylated.

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