title: An Efficient Trio-Based Mini-Haplotyping Method for Genetic Diagnosis of Phenylketonuria.
abstract: OBJECTIVE
  The phenylalanine hydroxylase (PAH) locus has high linkage disequilibrium. Haplotypes related to this locus may thus be considered sufficiently informative for genetic diagnosis and carrier screening using multi-allelic markers. In this study, we present an efficient method for haplotype analysis of PAH locus using multiplexing dyes. In addition, we explain how to resolve the dye shift challenge in multiplex short tandem repeat (STR) genotyping.
  
  
  METHODS
  One hundred family trios were included in this descriptive study. The forward primer of a tetra-nucleotide STR and the reverse primer of a variable number tandem repeat (VNTR) were labeled with three different non-overlapping dyes 5-carboxyfluorescein (FAM), 6-carboxy-N,N,N',N'-tetramethylrhodamine (HEX) and 6-carboxy-N,N,N',N'-tetramethylrhodamine (TAMRA). The polymerase chain reaction (PCR) products from each family trio were multiplexed for capillary electrophoresis and results were analyzed using Peak Scanner software.
  
  
  RESULTS
  Multiplexing trio products decreased the cost significantly. The TAMRA labeled products had a significant predictable shift (migrated at a slower electrophoretic rate) relative to the HEX and FAM labeled products. Through our methodology we achieve, the less inter-dye shift than intra-dye shift variance. Correcting the dye shift in the labeled products, according to the reference allele size, significantly decreased the inter-dye variability (P<0.001).
  
  
  CONCLUSIONS
  Multiplexing trio products helps to detect and resolve the dye shift accurately in each family, which otherwise would result in diagnostic error. The dye system of FAM, HEX and TAMRA is more feasible and cheaper than other dye systems.
authors.affiliation: Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Saeed
authors.initials: S
authors.lastname: Talebi
authors.affiliation: Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Mona
authors.initials: M
authors.lastname: Entezam
authors.affiliation: Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Neda
authors.initials: N
authors.lastname: Mohajer
authors.affiliation: Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Golnaz-Ensieh
authors.initials: GE
authors.lastname: Kazemi-Sefat
authors.affiliation: Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Masoumeh
authors.initials: M
authors.lastname: Razipour
authors.affiliation: Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Somayeh
authors.initials: S
authors.lastname: Ahmadloo
authors.affiliation: Department of Endocrinology, Children's Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Aria
authors.initials: A
authors.lastname: Setoodeh
authors.affiliation: Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Keramatipour
keywords: Capillary Electrophoresis; Diagnostic Error; Fluorescent Dyes; Microsatellite Repeats; Phenylalanine Hydroxylase