Detection of errors in dinucleotide repeat typing by nondenaturing electrophoresis.
This study shows that consideration of minor bands (heteroduplex, shadow, and faint bands) associated with allele bands in nondenaturing polyacrylamide gel electrophoresis (PAGE) after polymerase chain reaction (PCR) is effective for detecting PCR processing errors that lead to mistyping of heterozygotes as homozygotes. Notably, we show that minor bands in native gels are highly effective for detecting allele dropout and preferential amplification in PCR amplification of dinucleotide repeats. These findings are based on an analysis of Mendelian inheritance patterns in families, and the reproduction of heterozygous band patterns by mixing homozygous DNAs before PCR, for a total of six (AC)n repeats located on human chromosome 11p15. To investigate the utility of our approach, a large population sample of 405 unrelated individuals was genotyped for each (AC)n repeat using minor bands as internal quality controls. Genotype frequencies at each of the six loci were in close agreement with Hardy-Weinberg proportions, which suggests that there were few genotyping errors. Our observations add to the evidence indicating that minor bands in native gels are of diagnostic value in the genotyping of dinucleotide repeats.
Rodríguez
S
S
Departamento de Biología Fundamental, Universidad de Santiago, Santiago de Compostela, Spain.
Visedo
G
G
Zapata
C
C
D000180:Acrylic Resins; D000483:Alleles; D002880:Chromosomes, Human, Pair 11; D004247:DNA / Q000032:analysis*; D018900:Dinucleotide Repeats; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005838:Genotype; D006801:Humans; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods
D000180:Acrylic Resins; D009692:Nucleic Acid Heteroduplexes; C016680:polyacrylamide gels; D004247:DNA