RAPID SIZING OF SHORT TANDEM REPEAT ALLELES USING CAPILLARY ARRAY ELECTROPHORESIS AND ENERGY-TRANSFER FLUORESCENT PRIMERS

Genetic typing of the short tandem repeat (STR) polymorphism HUMTHO1 h as been performed using capillary array electrophoresis and energy-tra nsfer fluorescent dye-labeled polymerase chain reaction primers. Targe t alleles were amplified by use of primers labeled with one fluorescei n at the 5' end and another fluorescein at the position of the 15th (m odified) base to produce fragments that fluoresce in the green (lambda (max) = 525 nm). Unknown alleles were electrophoretically separated to gether with a standard ladder made up of alleles having 6, 7, 8, and 9 four-base pair repeats, each of which was amplified with an energy-tr ansfer primer having a donor fluorescein at the 5' end and a rhodamine acceptor at the position of the 7th (modified) base to produce standa rd fragments fluorescing in the red (>590 Mn). Separations were perfor med on arrays of hollow fused-silica capillaries filled with a replace able sieving matrix consisting of 0.8% hydroxyethyl cellulose plus 1 m u M 9-aminoacridine to enhance the resolution. The labeled DNA fragmen ts were excited at 488 nm, and the fluorescence was detected with a tw o-color confocal fluorescence scanner. Separations are complete in les s than 20 min and allow sizing with an average absolute error or accur acy of less than 0.4 base pair and an average standard deviation of si milar to 0.5 base pair with no correction for mobility shift and cross -talk between the fluorescence channels. This work establishes the fea sibility of high-speed, high-throughput STR typing of double-stranded DNA fragments using capillary array electrophoresis.