Detection of low abundant mutations in DNA using single-molecule FRET and ligase detection reactions

Abstract

New strategies for analyzing molecular signatures of disease states in real time using single pair fluorescence resonance energy transfer (spFRET) were developed to rapidly detect point mutations in unamplified genomic DNA (DNA diagnostics). The assay was carried out using allele-specific primers, which flanked the point mutation in the target gene fragment and were ligated using a thremostable ligase enzyme only when the genomic DNA carried this mutation (ligase detection reaction, LDR). We coupled LDR with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the MB probes formed upon ligation. We demonstrated the ability to rapidly discriminate single base differences in heterogeneous populations having as little as 600 copies of human genomic DNA without PCR amplification. Single base difference in the K-ras gene was discriminated in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA. Real time analyses of point mutations were also performed in PMMA microfluidic device.