Rapid p53 sequence analysis in primary lung cancer using an oligonucleotide probe array

Abstract

The p53 gene was sequenced in 100 primary human lung cancers by using direct dideoxynucleotide cycle sequencing and compared with sequence analysis by using the p53 GeneChip assay. Differences in sequence analysis between the two techniques were further evaluated to determine the accuracy and limitations of each method. p53 mutations were either detected by using both techniques or, if only detected by one technique, were confirmed by using mutation-specific oligonucleotide hybridization. Dideoxynucleotide sequencing of the conserved regions of the p53 gene (exons 5-9) detected 76% of the mutations within this region of the gene. The GeneChip p53 assay detected 81% of all (exons 2-11) mutations, including 80% of the mutations within the conserved regions of the gene. The GeneChip assay detected 46 of 52 missense mutations (88%), but 0 of 5 frameshift mutations. The specificity of direct sequencing and of the p53 GeneChip assay at detecting p53 mutations were 100% and 98%, respectively. The GeneChip p53 assay is a rapid and reasonably accurate approach for detecting p53 mutations; however, neither direct sequencing nor the p53 GeneChip are infallible at p53 mutation detection.

Figure 1

Schematic representation of chip array system. Probes on the array are arranged in sets of five. Each probe in the set is complementary to the reference sequence except for a mismatch position, called the “substitution” position. At the substitution position, each of the four possible nucleotides (A, C, G, T) and a single base pair deletion are represented in the probe set. Assay conditions optimize hybridization of the fluorescently labeled DNA target to the probe that best matches its sequence. This hybrid yields a higher fluorescence intensity relative to the other four target–probe hybrids in the set. There are probe sets complementary to every base in the p53 gene (figure courtesy of Affymetrix).

Figure 2

Comparison of direct dideoxynucleotide sequencing (a), mutation-specific oligonucleotide hybridization (b), and the p53 GeneChip assay (c and d) in sequence analysis of the p53 gene. Relative fluorescent intensity is shown for the sense (c) and antisense (d) oligonucleotide probe sets of the GeneChip but not for the 14 additional probe sets present at each of these base pairs. Sample 758 contains a missense mutation (cgc → ctc at codon 158) detected by all three techniques. Sample 1,140 contains the same mutation as sample 758 present as a faint band on the sequencing gel. However, it was not detected by the p53 GeneChip assay. A mutation at codon 253 in sample 1,049 was detected by the p53 GeneChip assay and confirmed by mutation-specific oligonucleotide hybridization despite its absence by direct sequencing.

Figure 3

Comparison of manual and automated sequence analysis. A CAC → CAT mutation is immediately evident on manual sequencing (Left) but is “missed” by automated analysis even after manual reading because of the very low height of the mutant T peak (red).