DIRECT SOLID-PHASE SEQUENCE-ANALYSIS OF THE HUMAN P53 GENE BY USE OF MULTIPLEX POLYMERASE CHAIN-REACTION AND ALPHA-THIOTRIPHOSPHATE NUCLEOTIDES

Among the candidate cancer-prognostic genes is the p53 tumor suppresso r gene, which, when mutated, plays an important role in the developmen t of many types of cancers. To facilitate robust large-scale DNA analy sis of microdissected tumor biopsies, we describe a multiplex/nested P CR approach for a simultaneous outer amplification of exons 4-9 of the human p53 gene with parallel amplification of the HLA-DQB1 locus, inv olving a total of 14 primers. This approach reduces the required numbe r of cells for analysis and avoids any variation in the amplifications of the individual p53 exons during the common outer amplification ste p. The HLA sequencing allows sample identification because the DQB1 lo cus is highly polymorphic and is thereby patient-specific. The p53 and HLA amplicons are analyzed by solid-phase sequencing in a semiautomat ed format. To improve the DNA sequence quality, we used 2'-deoxyribonu cleoside 5'-O-1-thiotriphosphates in the sequencing reactions.