AUTOMATED FLUORESCENT GENOMIC SEQUENCING AS APPLIED TO THE METHYLATION ANALYSIS OF THE HUMAN ORNITHINE DECARBOXYLASE GENE

A genomic sequencing method for an automated DNA sequencer was develop ed. The method described here is an improved version of the previously published protocol, which utilizes bisulfite-induced modification of genomic DNA. In our method, the modified DNA is purified without a tim e-consuming dialysis, and the subsequent 2-step DNA amplification is c arried out with one biotinylated primer in order to separate and isola te the strands of the product with the aid of streptavidin-coated magn etic beads. The strands are then sequenced with fluorescent primers an d automated DNA sequencer. This provides means to determine reliably t he methylation status of cytosines as well as the degree of methylatio n in a given CpG, site of the target sequence. The method was successf ully applied to analyze the promoter region and the 11th exon of the h uman ornithine decarboxylase ODC gene in various human myeloma cell li nes. The study revealed a totally unmethylated promoter region in ever y cell line studied, whereas the protein coding region appeared to be extensively methylated, although a dexamethasone resistant cell line d isplayed demethylation in certain CpG sequences. Also, a previously un known ODC allele was detected.