Detection of mutations by flow cytometric melting point analysis of PCR products

Abstract

Exploring the possibilities offered by flow cytometric microbead analyses for the detection of genetic alterations, an assay based on the dependence of the melting point of double-stranded DNA molecules on their length has been developed, making use of PCR products carrying biotin and fluorescent moiety on their two ends. The samples of different length PCR products immobilized on streptavidine coated microbeads are heat-treated in the presence of formamide at temperatures between the melting point of the longer and that of the shorter PCR product, when the mean fluorescence intensity of the beads carrying the shorter molecules decreases as a result of denaturation, as opposed to the sample containing the longer product. The efficacy and sensitivity of the method is demonstrated in the case of the assessment of the degree of triplet expansion in Huntington's disease. Its utility for the detection of point mutations in heterozygous clinical samples is shown in the case of the BRCA1 gene. The assay is simple and may be offered for the purposes of clinical diagnostics of a number of genetic conditions. These include screening of samples for triplet expansions and SNPs predisposing for particular pathological or pharmacogenomic conditions. In general, the method described herein is offered for the diagnosis of any pathological condition where the length of a genomic or cDNA sequence is expected to be different from that of the normal allele. IN conjunction with microbead analysis, flow cytometry is a sensitive and versatile platform for the biochemical, immunological, or molecular biological analysis of proteins and nucleic acids (1-9). Extending this spectrum, the method described herein is based on the dependence of the melting temperature [T m ; the temperature where 50% of the DNA molecules is present in denatured, single-stranded (ss) form] of double-stranded (ds) DNA molecules on their length. This temperature can be reduced to near ambient conditions in the presence of H-bond destabilizing reagents such as DMSO or formamide (10,11). Above its T m , a sample containing a PCR product prepared using a pair of biotinylated and fluorescent primers and immobilized on microbeads via the biotin moiety, will dissociate into free ss DNA molecules labeled with the fluorescent tag and the bead-attached non-fluorescent, biotinylated complementary strand. The degree of denaturation, that is, ratio of the ds and ss species at equilibrium will depend on the incubation temperature relative to the T m . Since the degree of denaturation at a given temperature is expected to depend also on the length of the DNA molecule (12,13), measurement of the above ratio by flow cytometry may be used for the length comparison of PCR products prepared from DNA samples in diseases where this approach might be of diagnostic value. The range of related potential applications include genetic disorders where the underlying disease might involve insertions, deletions, triplet expansions, microsatellite polymorphisms. In addition, when prim...