title: The effect of DNA labeling with the fluorescent dyes R110 and R6G on genotype analysis using capillary electrophoresis.
abstract: We investigated the mobility of DNA fragments labeled with the fluorescent dyes R110 and R6G, specifically for use in genotyping using capillary electrophoresis. Genomic DNA was isolated from blood, and a highly polymorphic region of the HLA-C gene was amplified by PCR with the use of either [F]-dNTP-[R110] or [F]-dNTP-[R6G]. Pre-diluted (30-fold) PCR products were mixed with formamide, denatured (at 95 degrees C for 2 min.), and rapidly cooled on ice before being subjected to electrophoresis. The results showed that the number and mobility of allele-specific DNA fragments were independent of the two dyes used. Both dyes were equally efficient at differentiating homozygous or heterozygous allelic presentation. An additional dye-specific peak of 132-base-pair mobility was observed with the use of [F]-dNTP-[R110]; it significantly impaired the resolution of one allele-specific peak. The electropherograms obtained with [F]-dNTP-[R6G] were free from any interfering peaks within the target region, thus the [R6G]-based procedure is more preferable for genotype analysis. As this procedure does not involve any post-PCR cleanup, it is simple, rapid and cost-effective.
authors: Department of Biochemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia. haseeb@ksu.edu.sa
authors: Haseeb Ahmad
authors: HA
authors: Khan
mesh_terms: D000483:Alleles; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D005838:Genotype; D015236:HLA-C Antigens / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction; D012235:Rhodamines / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D015236:HLA-C Antigens; D012235:Rhodamines; D004247:DNA