Detection of the XXXX mutation by a modified photo-cross-linking oligonucleotide hybridization assay.

@entity5337 @entity21801 @entity941
BACKGROUND: Our previously developed assay for detection of the @entity5337 mutation ( @entity21801 ) based on a nucleic acid photo-cross-linking technology used two allele-specific capture probes and six @entity941 -modified signal-generating reporter probes. We wished to improve the sensitivity and performance of the method. METHODS: We developed new reporter probes with approximately 10-fold more @entity941 molecules than the original probes. The single, cross-linker-modified capture probe was replaced by a three-probe system, separating the probe-target cross-linking function and the allelic differentiation function. The capture probe cross-linked to either or both of two flanking probes through stem structures at the capture-probe/flanking-probe junctions. The flanking probes cross-linked to target DNA through two cross-linking sites each. Genomic DNA was extracted from 0.2 mL of whole blood and restriction-enzyme digested to create a defined 677 bp target sequence. Preliminary genotype ranges were determined for the assay by testing of pre-typed samples. We then tested 1054 clinical samples, using an automated sample processor. RESULTS: The new assay had a 10-fold increase in signal-to-background ratio. Genotype results for 1039 of 1054 clinical samples (98.6%) agreed with those of a PCR-based method. Of the 15 remaining samples, 10 produced an indeterminate result outside the defined genotype ranges, 2 yielded insufficient signal to be genotyped, and 3 gave a discordant result. All 15 samples were genotyped correctly after re-extraction of genomic DNA and retesting. CONCLUSION: The modified photo-cross-linking assay for @entity5337 detection is a sensitive non-PCR-based assay with potential for use in high-throughput clinical laboratories.
