Evaluation of the arrayed primer extension resequencing assay for TP53mutation detection

Over the years we have screened for TP53 mutations in different patient materials using temporal temperature gel electrophoresis (TTGE) [1], followed by direct sequencing of samples with aberrant migrating bands to determine the nature of the sequence alteration. Mutations in the TP53 gene are associated with several different cancer types and have been shown to have both prognostic and predictive implications. In this project we are evaluating whether a commercial available array platform for sequencing the TP53 gene using a primer extension assay (APEX) is as sensitive, rapid and cost-effective as TTGE/sequencing.

The array is designed by Asper Biotech [2]. Genomic DNA is amplified by PCR, and dUTP is incorporated. The amplification products are then concentrated and purified with spincolumns. Amplification products are fragmented by Uracil N-glycosylase, and unincorporated dNTPs are inactivated by shrimp alkaline phosphatase. The fragmented PCR products are mixed with thermosequenase and four fluorescence-labelled ddNTPs. The sample mixture is transferred to a chip that contains sequence-specific oligonucleotides. So far, exons 2–9 are included on the array. Genorama™ QuattroImager is used for scanning. The Genorama imaging system and genotyping software are used for imaging and semiautomatic sequence analysis.

DNA samples from 48 primary breast carcinomas, 11 ovarian carcinomas and 34 cell lines were used for evaluation. Results from a titration experiment with different ratios of the Arg/Arg and Pro/Pro alleles on codon 72 in the TP53 gene showed that mutations could be detected even if the mutated cells were present in less than 5%. We have experienced that homozygous and hemizygous mutations occasionally are missed by the TTGE technique, but that they all were easily detected by APEX [3]. Detection of deletions and insertions, however, is not yet optimal using the APEX technology and they are frequently missed. For the tumour samples the resequencing efficiency using APEX was 92% for both DNA strands and 99.5% for sense and/or antisense strands.

The strength of using the APEX technology is that both strands are simultaneously analyzed, and that no further sequencing is needed. It is rapid and sensitive. Cost-effectiveness is still under evaluation.