Energy-transfer cassette labeling for capillary array electrophoresis short tandem repeat DNA fragment sizing

Energy-transfer (ET) dye-labeled primers significantly improve fluorescent DNA detection because they permit excitation at a single common wavelength and they produce well separated and intense acceptor dye emission. Recently , a new ET cassette technology was developed [Berti, L. et al. (2001) Anal. Biochem. 292, 188-197] that can be used to label any PCR, sequencing, or o ther primer of interest. In this report we examine the utility of this ET c assette technology by labeling seven different short tandem repeat (STR) sp ecific primers with each of the four ET cassettes and analyzing the PCR pro ducts generated on a MegaBACE-1000 capillary array electrophoresis system. More than 60 amplicons were generated and successfully analyzed with the ET cassette-labeled primers. Both forward and reverse primers were labeled fo r multiplex PCR amplification and analysis. Single base pair resolution was achieved with all four ET cassettes. This ET cassette-primer labeling proc edure is ideally suited for creating four-color fluorescent ET primers for STR and other DNA assays where large numbers of different loci are analyzed including sequencing, genetic identification, gene mapping, loss of hetero zygosity testing, and linkage analysis.