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 other primer of interest. In this report we examine the utility of this ET cassette technology by labeling seven different short tandem repeat (STR) specific primers with each of the four ET cassettes and analyzing the PCR products 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 for multiplex PCR amplification and analysis. Single base pair resolution was achieved with all four ET cassettes. This ET cassette-primer labeling procedure 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 heterozygosity testing, and linkage analysis.
Berti
L
L
Department of Chemistry, University of California, Berkeley, California 94720, USA.
Medintz
I L
IL
Tom
J
J
Mathies
R A
RA
D004247:DNA / Q000032:analysis* / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019075:Electrophoresis, Capillary; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D020014:K562 Cells; D016133:Polymerase Chain Reaction; D020080:Tandem Repeat Sequences / Q000235:genetics*
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA