The effect of DNA labeling with the fluorescent dyes R110 and R6G on genotype analysis using capillary electrophoresis.
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.
Khan
Haseeb Ahmad
HA
Department of Biochemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia. haseeb@ksu.edu.sa
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
D005456:Fluorescent Dyes; D015236:HLA-C Antigens; D012235:Rhodamines; D004247:DNA