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 developed. 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 time-consuming dialysis, and the subsequent 2-step DNA amplification is carried out with one biotinylated primer in order to separate and isolate the strands of the product with the aid of streptavidin-coated magnetic beads. The strands are then sequenced with fluorescent primers and automated DNA sequencer. This provides means to determine reliably the methylation status of cytosines as well as the degree of methylation in a given CpG, site of the target sequence. The method was successfully applied to analyze the promoter region and the 11th exon of the human ornithine decarboxylase ODC gene in various human myeloma cell lines. The study revealed a totally unmethylated promoter region in every cell line studied, whereas the protein coding region appeared to be extensively methylated, although a dexamethasone resistant cell line displayed demethylation in certain CpG sequences. Also, a previously unknown ODC allele was detected.