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Glal hydrolysis and Ligation Adapter Dependent PCR assay is the novel method to determine RGY sites produced in the course of de novo DNA methylation with DNMTЗA and DNMTЗB DNA methyltransferases. GLAD-PCR assay do not require bisulfite treatment of the DNA.
Method was specially designed to determine methylation of RCGY site of interest in human and mammalian genomes in excess of corresponding unmethylated sites. This is a typical situation for DNA preparations from clinical samples of blood and tissues.
GLAD-PCR assay is based on the new type of enzymes - site-specific methyl-directed DNA-endonucleases. These enzymes are very similar to restriction enzymes in biochemical properties and cleave DNA completely, but act in opposite way: they cleave only methylated DNA and do not cleave unmethylated DNA at all.
Mammalian DNA-methyltransferases DNMT1, DNMT3a and DNMT3b catalyze a reaction of DNA methylation.