Direct sequencing from touch preparations of human carcinomas: analysis of p53 mutations in breast carcinomas.

A new technique for characterizing somatic mutations in very small samples of cellularly heterogeneous human cancer tissue was developed and tested using mutations in the p53 gene in breast carcinomas as a model system. The technique combines touch preparation of specimens to obtain homogeneous clusters of carcinoma cells free of normal cells with a nested pair of polymerase chain reaction (PCR) amplifications of DNA to increase the amount of target gene sequence sufficiently to permit direct sequencing of the p53 gene. Touch preparations of fresh or previously frozen tissue from human adenocarcinomas derived from several organs were stained, and clusters of 10-50 malignant cells were transferred by pipette into microfuge tubes for PCR amplification. Exons 5-9 of the p53 gene, which contain the major mutational hot spots associated with most human cancers, were sequenced by the following steps: 1) two rounds of PCR amplification using DNA Taq polymerase and two sets of oligonucleotide primers, the second set being nested within the segment amplified by the first set and having attached T7 and SP6 phage promoter sequences, 2) transcription of the amplified DNA sequences with T7 and SP6 RNA polymerases, and 3) dideoxy sequencing of single-stranded RNA transcripts with reverse transcriptase and with additional oligonucleotide primers to achieve specificity for this unique region of the genome. The utility of this approach is illustrated by our success in detecting and analyzing point mutations in cell clusters from four of 11 primary adenocarcinomas of the human breast.

A novel method for detecting point mutations or polymorphisms and its application to population screening for carriers of phenylketonuria.

We describe a method termed PCR (polymerase chain reaction) amplification of specific alleles (PASA), a generally applicable technique for detection of point mutations or polymorphisms. The ease and technical simplicity of PASA will make genetic analyses more accessible to the general medical community. In addition, PASA shows promise for population screening because the technique is rapid, highly reproducible, inexpensive, nonisotopic, and amenable to automation. PASA is a modification of PCR that depends on the synthesis of a PCR oligonucleotide primer that precisely matches with one of the alleles but mismatches with the other. When the mismatch occurs near the 3' end of the PCR primer, amplification is inefficient. Therefore, preferential amplification of the perfectly matched allele is obtained. We demonstrate the applicability of PASA by performing carrier detection in the family of a patient with phenylketonuria (PKU) and by screening a population of unrelated subjects for the presence of the two mutations most commonly associated with PKU. Multiple persons were screened simultaneously for the mutant alleles because a mutation could be detected in the presence of at least a 40-fold excess of the normal allele. The two PKU mutations could be detected concurrently by using a mixture of only three PCR primers, an indication that simultaneous screening of multiple mutations can be done even if three or more mutations are closely clustered. In addition to the detection of mutations, PASA can be used to detect polymorphic alleles rapidly and to distinguish pseudogenes or repetitive sequences that differ by as little as one base.