Fluorescent resonance energy transfer (FRET) based detection of a multiplex ligation-dependent probe amplificationassay (MLPA) product

Abstract

A fluorescentresonance energy transfer (FRET)-based hybridizationassay for detecting multiplex ligation-dependent probeamplification (MLPA) products has been developed, extending the diagnostic power of the technique and demonstrating the possibility of combining MLPA with microarrays for the detection of multiple mutations. FRET is one of the most commonly used detection techniques for hybridizationassays. To investigate the applicability of FRET based detection of MLPA products, a sandwich assay was designed to detect gene copy number by exploiting an immobilized probe labeled with an acceptor dye, Alexa Fluor 555, which hybridises to specific PCR amplicons, followed by hybridization of a second probe labeled with the donor dye, Alexa Fluor 488. Following excitation of the Alexa Fluor 488, a FRET signal was produced only if a DNA sequence specific to the BRCA1exon 13 was present in the test sample. We have verified this assay on a DNA sample of a patient carrying a heterozygous BRCA1exon 13 deletion using male genomicDNA as control. Here we demonstrate that the DNA sample containing the heterozygous deletion generated a considerably reduced FRET signal as compared to the control male human DNA. Our results show that the FRET design presented in this study can differentiate between reduced copy numbers any genomicDNA sequence after MLPA analysis, and the reported format is applicable to multiplex detection of MLPA products, using microarrays, or optical biosensor arrays, and future work will focus on the demonstration of this.