Analysis of the D1S80 locus by capillary electrophoresis.
We have demonstrated in a systematic manner that the capillary electrophoresis (CE) method of genotyping the human D1S80 locus is an effective replacement for the commonly used gel electrophoresis method. The CE method is fast, with a total run time of less than 22 min per sample. Separation has been optimized so that resolution for all pairs of alleles from 14 through 41 is greater than 1, providing unambiguous identification. The method utilizes run buffer containing 0.30% hydroxyethyl cellulose as the sieving polymer in a 50 microm internal diameter (ID) column coated with a 0.1 microm DB-17 film. Laser-induced fluorescence (LIF) with YO-PRO-1 intercalating dye is used for detection. Internal standards of 300 and 1000 bp bracket the D1S80 region in the electropherogram. Two typing procedures were evaluated: matching of the normalized migration times of sample alleles to ladder alleles; and software alignment of sample and ladder electropherograms using the internal standard peaks as references. Using the first procedure, 91.5% of the D1S80 genotypes were unambiguous, while 100% were unambiguous using the second procedure. Seventy-nine routine samples were analyzed in a side-by-side comparison of the CE and slab gel methods, with complete agreement of the results. Twenty-two samples were selected from a large database previously analyzed by the slab gel method to demonstrate that all alleles from 14 through 41 were typed correctly, including samples containing adjacent alleles. Additionally, 43 samples containing alleles greater than the 41 repeat number were typed correctly.
Mitchell
J W
JW
IIT Research Institute, Newington, VA 22122, USA. jmitchel@vtc.iitri.com
Walsh
D
D
D001331:Automation; D002874:Chromosome Mapping; D019075:Electrophoresis, Capillary / Q000379:methods*; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction