Typing of the short tandem repeat D8S347 locus with different fluorescence markers

Abstract

The short tandem repeat (STR) locus D8S347 was analyzed by capillary electrophoresis. Sequencing data and a population study of 203 individuals from a southwestern German population are presented. We detected 12 different alleles, 340-388 bp in length, and found 40 different genotypes. The heterozygosity index was 85.7%. Futhermore, we investigated the consequences of different fluorescent dyes, namely 6-FAM, HEX, and ROX, on the ABI-calculated fragment sizes of defined (i.e., sequenced) alleles (348-376 bp in length). 6-FAM-labeled fragments appear to be smaller than the corresponding HEX- or ROX-labeled fragments. On average, 6-FAM-labeled fragments differ by 3.52 bp from the sequencing data, HEX-labeled ones by 2.04 bp, and ROX-labeled ones by 1.42 bp. Generally, small alleles differ less from the expected sequencing data than larger ones.

Influence of fluorophor dye labels on the migration behavior of polymerase chain reaction - amplified short tandem repeats during denaturing capillary electrophoresis

“…The discrepancy is due to the latter method which leads to false determinations of genotypes as was shown with sequence-verified STR fragments. Similar effects were described by Poltl et al [8] for the STR marker D8S347 though these authors used a different polymer and other run conditions and found other trends in mistyping.…”

“…The classical method for identification of STR alleles will remain the method-of-choice: the spiking of sample with an aliquot of an allele ladder of this STR marker, labelled with the same fluorophor dye [8,24]. A compromise might be to use different dyes of similar physicochemical properties and migration behavior (e.g., only fluorescein derivatives).…”

“…In addition, the development of dedicated instruments, equipped with multichannel laserinduced fluorescence (LIF) detection (e.g. ABI PRISM 310 Genetic Analyzer, Applied Biosystems [1]), facilitated the acceptance and widespread use of this method [1][2][3][4][5][6][7][8][9].…”

Accurate and sensitive analysis of triplet repeat expansions by capillary electrophoresis

“…The difference in the type of the DNA sequence between the analyzed sample and coelectrophoresed DNA size standard is one of the factors responsible for the reported size estimation errors and discordant results reported by different authors [6,8,12]. Although the allelic ladders were proposed to eliminate this problem [13,14] the differences in migration rates between DNA fragments having the same sequence but labeled with different fluorochromes still remained [18,22,23]. The approach we propose for a reliable CAG repeat length analysis is free of the above drawbacks.…”

“…For example, the p53 gene fragments 5'-labeled with fluorescein derived dyes (JOE, 6-FAM, FAM, NED, TET, HEX) appeared to be smaller by up to 6.5 bp than the actual fragment size [22]. According to another report the DNA fragments labeled with 6-FAM differed by 3.52 bp, the HEX-labeled fragments of the same length and sequence differed by 2.04 bp, and the same fragments labeled with ROX differed by 1.42 bp from the sequencing results [23].…”