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Influence of fluorophor dye labels on the migration behavior of polymerase chain reaction – amplified short tandem repeats during denaturing capillary electrophoresis
Author(s) -
Hahn Meinhard,
Wilhelm Jochen,
Pingoud Alfred
Publication year - 2001
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200108)22:13<2691::aid-elps2691>3.0.co;2-s
Subject(s) - capillary electrophoresis , genotyping , chemistry , oligonucleotide , tandem repeat , polymerase chain reaction , dna , electrophoresis , polyacrylamide , rhodamine , fluorescein , gel electrophoresis , fluorescence , chromatography , microbiology and biotechnology , biology , genotype , gene , biochemistry , polymer chemistry , physics , quantum mechanics , genome
The determination of the length of polymerase chain reaction (PCR)‐amplified short tandem repeats (STRs) by denaturing capillary electrophoresis (CE) is a standard procedure for purposes of genotyping. We show that dye‐specific mobility anomalies exist for 5′‐fluorophor‐labelled single‐stranded DNA (ssDNA) fragments in CE using the performance‐optimized polymer 4 (POP4) buffer sieving matrix, containing the entangled poly( N,N ‐dimethylacrylamide) polymer, urea, and 2‐pyrrolidinone. The dye‐specific retardation effects relative to coseparated Gene Scan‐500 [TAMRA] standard fragments can lead to wrong genotyping, even for allele‐specific fragments of pentanucleotide STRs, when comparing the relative calculated sizes of identical fragments, labelled with rhodamine (ROX, TAMRA) or fluorescein dyes (FAM, 6‐FAM, HEX, JOE, NED, TET): The size of fluorescein dye‐labelled fragments of appr. 100 b in length appears to be smaller by up to 6.5 b. This effect becomes more dramatic with decreasing size: a 6‐FAM‐labelled 24‐mer oligonucleotide appeared to be smaller by 11 b. In contrast, in classical urea/polyacrylamide slab‐gel electrophoresis only a small dye‐specific retardation of identical fragments is observed. The dye‐specific effects are superimposed by weaker size and sequence‐dependent anomalies of fragment mobility. Therefore, in denaturing CE the coseparation of a defined allele ladder labelled with the same dye as the unknown sample fragments remains the method of choice for accurate genotyping.