title: Clinical applications of denaturing high-performance liquid chromatography-based genotyping.
abstract: OBJECTIVE
  To develop and evaluate heteroduplex forming templates (HFTs) as a common set of molecular standards for genotyping by denaturing high-performance liquid chromatography (DHPLC) using hypervariable regions of human mitochondrial DNA (mtDNA) as a model system.
  
  
  METHODS
  Hypervariable regions 1 and 2 from the mtDNA D-loop of 22 maternally related and unrelated human volunteers were amplified by polymerase chain reaction (PCR) and individually mixed with each of three HFTs. Following denaturation and reannealing of the mixture, the resulting hetero- and homoduplicies were separated by DHPLC using temperature-modulated heteroduplex analysis.
  
  
  RESULTS
  Each of three HFTs, when cross-hybridized with a target mtDNA amplicon, induced the formation of an assemblage hetero- and homoduplex peaks, which were uniquely characteristic of a given mtDNA sequence variant. The mtDNA DHPLC profiles obtained in the current study were identical between maternal relatives and different between unrelated individuals--consistent with uniparental maternal inheritance of mtDNA in humans.
  
  
  CONCLUSIONS
  DHPLC in combination with a common set of HFTs targeted to a locus of interest can be used as a reliable means of genotyping. DHPLC profiles can be readily stored as a bit-coded string of hetero- and homoduplex peak retention times to form a searchable database. This approach to DHPLC genotyping will have immediate utility in extended pedigree analyses, where it will allow rapid sorting and/or confirmation of maternal lineages. Additional applications of DHPLC profiling include the discovery and scoring of clinically relevant nuclear and mitochondrial loci.
authors.affiliation: University of Denver, Department of Biological Sciences, 2101 E. Wesley Avenue Rm. 211, Denver, Colorado 80208, USA. pdaniels@du.edu
authors.forename: Phillip B
authors.initials: PB
authors.lastname: Danielson
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Shelton
authors.forename: Greggory S
authors.initials: GS
authors.lastname: LaBerge
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D004272:DNA, Mitochondrial / Q000235:genetics*; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012016:Reference Values; D012494:Sampling Studies; D012680:Sensitivity and Specificity
chemical_list: D004272:DNA, Mitochondrial