Short tandem repeat typing by capillary array electrophoresis: comparison of sizing accuracy and precision using different buffer systems.
Polymorphic microsatellite markers are widely used in gene discovery and mapping, human identification, agricultural genetics, and diagnosis of triplet-repeat expansion disorders. Reliable genotyping of these markers requires polymerase chain reaction (PCR) amplification and very-high-resolution electrophoresis. Capillary array electrophoresis offers extremely fast, high-resolution separation of DNA and more automated sample processing because labor-intensive slab-gel pouring and sample loading are eliminated. We report a simple, reliable procedure for preparing PCR samples for electrokinetic injection into capillaries using a 96-well tray and float dialysis. We developed an improved sizing standard for genotyping and used it to evaluate systematically the sizing accuracy and precision of low-viscosity, replaceable matrix formulations. Our study sizing over 28,000 alleles yielded an average precision of +/- 0.12 bp for fragments up to 350 bp. Low-viscosity formulations permit low-pressure matrix injection (40 psi) and a turnaround time of 70 min for 48-96 samples.
Vainer
M
M
Molecular Dynamics, Inc., Sunnyvale, California 94086, USA.
Enad
S
S
Dolnik
V
V
Xu
D
D
Bashkin
J
J
Marsh
M
M
Tu
O
O
Harris
D W
DW
Barker
D L
DL
Mansfield
E S
ES
D000483:Alleles; D001483:Base Sequence; D002021:Buffers; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004586:Electrophoresis / Q000379:methods* / Q000592:standards; D005069:Evaluation Studies as Topic; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D014783:Viscosity
D002021:Buffers; D004247:DNA