RAPID SEQUENCING OF THE P53 GENE WITH A NEW AUTOMATED DNA SEQUENCER

p53 is the most commonly mutated gene in human cancers. Approximately 90% of the p53 gene mutations are localized between domains encoding e xons 5 to 8. Sequencing methods currently available are tedious and ti me-consuming and are not suitable for routine laboratory testing. In a n effort to identify a simple and rapid sequencing method, we analyzed 16 preselected breast tumors and 18 preselected ovarian tumors, using at newly developed automated DNA sequencer. p53 gene mutations had be en previously identified in these tumors, using a conventional automat ed sequencing procedure. Exons 5 to 8 were amplified by PCR, and the P CR products were subsequently subjected to cycle sequencing with the S anger chain termination method, using Cy5.5-labeled primers. The seque ncing mixture was then resolved on a newly developed automated DNA seq uencer that can sequence similar to 300 bases of DNA in 30 min. Of the se 16 breast tumors, two had mutations in exon 5, four in exon 6, thre e in exon 7, and three in exon 8. Of the 18 ovarian tumors, two had mu tations in exon 5, five in exon 6, two in exon 7,and three in exon 8. In all cases, we identified the same mutations by both the new and the conventional sequencing procedures. Most mutations affected an argini ne codon. These data demonstrate that the new method has the capabilit y to provide accurate sequencing information in a fraction of the time and labor in comparison with current auto mated sequencing techniques . When such procedures are used, DNA sequencing may become a routine t ool for identifying clinically important mutations far diagnosis and p rognosis of patients with genetic, malignant, infectious, and other di seases.