Combinatorial fluorescent energy transfer tags and their application for multiplex genetic analyses

Combinatorial fluorescent energy transfer (CFET) tags should be valuable for multiplex genetic mutation analysis, DNA mapping, genomewide chromosome analysis as well as other multi-component analysis systems. As an example, we present the use of CFET tag for multiplex genetic mutation detection using ligase chain reaction. Ligase chain reaction (LCR) is a procedure for genetic mutation analysis using ligase and a pair of oligonucleotides spanning the ligation site on the template DNA, including one harboring either the wild-type or mutated base (Eggerding, 1995). In this procedure, one of the oligonucleotides is fluorescently labeled at its 5' end. Following the ligase chain reaction, which involves multiple rounds of denaturing, primer annealing and ligation, one can identify the ligated products on polyacrylamide gels. We have applied CFET tag labeled oligonucleotides to ligase chain reaction that allows the simultaneous analysis of a large number of genetic mutations in a single tube reaction. For example, we can identify all the known single base mutations in exon 20 of the retinoblastoma susceptibility (Rbl) gene using eight CFET tags constructed from three individual fluorophores.