Evaluating the arrayed primer extension resequencing assay of TP53 tumor suppressor gene

Abstract

Identification of mutations in the tumor suppressor gene TP53 has implications for the molecular epidemiology and for the molecular pathology of human cancer. We have developed and evaluated an arrayed primer extension assay for covering both strands of a region of the coding sequence containing more than 95% of the mutations described so far in TP53. On average, 97.5% of the arrayed TP53 gene sequence can be analyzed from either sense or antisense strands, and 81% from both strands. A patient DNA sample is amplified and annealed to arrayed primers, which then promote DNA polymerase extension reactions with four fluorescently labeled dideoxynucleotides. The TP53 gene chip spans exons 2-9 plus two introns from both strands. The performance of the assay was evaluated by using freshly extracted genomic DNA, as well as DNA extracted from archival (paraffin-embedded) DNA samples. The arrayed primer extension-based TP53 gene test provides an accurate and efficient tool for DNA sequence analysis of this frequently mutated gene for both research and clinical applications.APEX ͉ oligonucleotide array ͉ chip T he evidence is growing that specific mutations in the TP53 gene can represent important factors for the prognosis of cancer and for the response to various types of cytotoxic therapy. Furthermore, patterns of TP53 mutations have differed considerably from one type of cancer to the other (1-4). However, screening for TP53 mutations gene has yet to become a routine in clinical or epidemiological practice, mainly because current detection technologies are labor-intensive and have prohibitive costs for large-scale prospective studies. Another strong limitation to routine analysis of TP53 mutations resides in the fact that many tumors contain an excess of wild-type TP53 as compared with mutant, resulting from the presence of intact alleles in tumor as well as in noncancer cells (stroma, inflammatory cells, blood vessels).In this report we describe the development of an arrayed primer extension (APEX) assay for the rapid and sensitive detection and identification of mutations in the TP53 gene. APEX is a genotyping and resequencing technology that combines the advantages of Sanger dideoxy sequencing with the parallelization and high-throughput potential of the microarray format. A DNA sample is amplified, fragmented enzymatically, and annealed to arrayed primers, promoting sites for templatedependent DNA polymerase extension reactions by using four fluorescently labeled dideoxynucleotides. Each base is probed with two primers, one for the sense and another for the antisense strand (5). GENORAMA imaging system and genotyping software (Asper Ltd., Tartu, Estonia, www.asperbio.com) were used for imaging and semiautomatic sequence analysis (Fig. 1).The principle of sequencing by primer extension on oligonucleotide array has been successfully applied for the systematic identification of all common TP53 mutations in human cancers. The TP53 microarray presented here spans exons 2-9 [containing more than 98% of all mutations descr...

A TP53 polymorphism is associated with increased risk of colorectal cancer and with reduced levels of TP53 mRNA

“…All subjects gave written informed consent according to the Helsinki declaration. The p53PIN3 variant was genotyped as previously reported (Lazar et al, 1993), while the Arg72Pro variant was analysed using a primer extension approach (To˜nisson et al, 2002). The MspI intron 6 polymorphism was not investigated because it has been reported to be in complete linkage disequilibrium (LD) with the p53PIN3 variant (Weston et al, 1997).…”

Detection of single nucleotide polymorphisms by minisequencing on a polypyrrole DNA chip designed for medical diagnosis

“…This compares favorably with the sensitivity observed by using minisequencing and fluorescence detection on a glass support. 42 For mutation genotyping, this detection threshold could be lowered. One option is to reduce the noise level by modifying the optics of the Apimager s .…”

“…This length is similar to those previously reported in this type of array assay. 42 For liquid-phase minisequencing, 24-to 30-base-long SNP primers should be used for experiments based on fluorescence polarization, whereas for mass spectroscopy assays, probes as short as 16 bases are preferred. In the absence of a cycling reaction, allowing the generation of large amounts of extension products, the quality of the target is primordial.…”

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

“…Technically, polymorphisms are identified by their difference in duplex stability on a set of oligonucleotides that exhibit all possible sequence variations. Alternatively, discrimination is achieved by the specificity of a polymerase, incorporating the respective nucleotide at the position of interest (e.g., [114]). …”