General description

Methylation of cytosines located 5′ to guanosine is known to have a profound effect on the expression of many eukaryotic genes. In normal cells methylation occurs predominantly in CG-poor regions, while CG-rich areas, called CpG-islands remain unmethylated. The exceptions are the extensive methylation of CpG islands associated with transcriptional inactivation of regulatory regions of imprinted genes and genes on the inactive X-chromosome of females. Aberrant methylation of normally unmethylated CpG islands has been documented as a relatively frequent event in immortalized and transformed cells and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers.

Several methods have been developed to determine the methylation status of cytosine. These include the use of antibodies or protein methyl binding domains, digestion with methylation sensitive, insensitive, or dependent restriction enzymes as in restriction landmark genomic scanning (RLGS), oligonucleotide array hybridization, bisulfite genomic DNA sequencing and Methylation Specific PCR (MSP).

Genomic DNA sequencing, although time consuming and labor intensive, offers a more universal detection method. MSP is an established technology for the monitoring of abnormal gene methylation in selected gene sequences. Utilizing small amounts of DNA, this procedure offers sensitive and specific detection of 5-methylcytosine in promoters. It is being exploited to define tumor suppressor gene function, and to provide a new strategy for early tumor detection by interrogating DNA derived from tissues and bodily fluids.

The initial step of both bisulfite genomic sequencing and MSP is to perform a bisulfite modification of the DNA sample. In the bisulfite reaction, all unmethylated cytosines are deaminated and sulfonated, converting them to uracils, while 5-methylcytosines remain unaltered. Thus, the sequence of the treated DNA will differ depending on whether the DNA is originally methylated or unmethylated. Also, the initially complementary DNA strands will no longer be complementary after cytosine conversion. Primers for use in MSP can be designed to specifically amplify either a bisulfite-sensitive, unmethylated strand or a bisulfite-resistant, methylated strand, based upon these chemically-induced differences. Millipore offers a selection of CpG Wiz MSP kits to enable gene-specific analysis by MSP. To learn more about CpG Wiz kits and MSP technology Click here

The CpGenome Turbo Bisulfite Modification Kit is designed to simplify and streamline the bisulfite modification process. This kit contains all key reagents for bisulfite modification to allow for the recovery of modified DNA in about 90 minutes as compared to 3-16 hours for similar kits. This rapid, but effective approach employs a proprietary mixture of modification reagents to permit shorter conversion times. This unique mixture of reagents allows for complete conversion of unmethylated cytosines while minimizing damage to the input DNA. After modification, and desulfonation, the modifed DNA is purified and eluted using the provided spin columns resulting in purified, bisulfite-modifed DNA that is ready to use in a wide variety of downstream applications.

Key Features

Input sample to modified DNA in 90 minutes
Conversion efficiencies of 99.9%
Effective with as little as 1 ng input DNA
Recover modified DNA in as little as 25 microliters
Proven performance in multiple downstream applications

Application

For MSP primer design, please use the MethPrime software package. Click here

Research Category
Epigenetics & Nuclear Function

Components

Bisulfite Conversion Reagent 1

Bisulfite Conversion Reagent 2

Bisulfite Reagent Diluent

DNA Binding Buffer

Wash Buffer NT3 (Concentrate)*

Elution Buffer NE

Modified DNA Purification Columns

2 mL Collection Tubes

*DNA Wash Buffer requires the addition of 100% Ethanol

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.