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 has been performed using capillary array electrophoresis and energy-transfer fluorescent dye-labeled polymerase chain reaction primers. Target alleles were amplified by use of primers labeled with one fluorescein at the 5' end and another fluorescein at the position of the 15th (modified) base to produce fragments that fluoresce in the green (lambda max = 525 nm). Unknown alleles were electrophoretically separated together 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-transfer primer having a donor fluorescein at the 5' end and a rhodamine acceptor at the position of the 7th (modified) base to produce standard fragments fluorescing in the red (> 590 nm). Separations were performed on arrays of hollow fused-silica capillaries filled with a replaceable sieving matrix consisting of 0.8% hydroxyethyl cellulose plus 1 microM 9-aminoacridine to enhance the resolution. The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two-color confocal fluorescence scanner. Separations are complete in less than 20 min and allow sizing with an average absolute error or accuracy of less than 0.4 base pair and an average standard deviation of approximately 0.5 base pair with no correction for mobility shift and cross-talk between the fluorescence channels. This work establishes the feasibility of high-speed, high-throughput STR typing of double-stranded DNA fragments using capillary array electrophoresis.
Wang
Y
Y
Department of Chemistry, School of Public Health, University of California, Berkeley 94720.
Ju
J
J
Carpenter
B A
BA
Atherton
J M
JM
Sensabaugh
G F
GF
Mathies
R A
RA
D000483:Alleles; D000585:Aminacrine; D001483:Base Sequence; D004247:DNA / Q000032:analysis; D017931:DNA Primers; D004586:Electrophoresis / Q000379:methods; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D008969:Molecular Sequence Data; D010316:Particle Size; D016133:Polymerase Chain Reaction / Q000379:methods; D012091:Repetitive Sequences, Nucleic Acid; D013194:Staining and Labeling / Q000379:methods; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
D017931:DNA Primers; D005452:Fluoresceins; D005456:Fluorescent Dyes; C045132:4-carboxyfluorescein; D000585:Aminacrine; D004247:DNA; D014446:Tyrosine 3-Monooxygenase