title: Molecular haplotyping by linking emulsion PCR: analysis of paraoxonase 1 haplotypes and phenotypes.
abstract: Linking emulsion PCR (LE-PCR) enables formation of minichromosomes preserving phase information of two polymorphic loci, hence the haplotype. Emulsion PCR confines two amplicons of two linked polymorphic sites on a single template molecule to one aqueous-phase droplet. Linking PCR uses biotinylated, overlapping linking primers to connect these amplicons in the droplet. After LE-PCR, unlinked amplicons are removed on streptavidin-coated magnetic beads and single-stranded runoff products are capped by primer extension. Quantitative ASPCR can then be used to ascertain the haplotypes of the two polymorphic loci on the minichromosomes. Using LE-PCR, we determined the human paraoxonase-1 [PON1] molecular haplotypes at three loci (-909g>c, L55M, Q192R) in women who were compound heterozygotes for -909g>c/L55M (n = 89), -909g>c/Q192R (n = 77) and L55M/Q192R (n = 68). We observed a strong association between PON1 substrate specificity (paraoxon/phenylacetate substrate activity ratios) and -909g>c/Q192R haplotype. We have demonstrated here a powerful molecular haplotyping technology that can be applied in population studies.
authors.affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA. james.wetmur@mssm.edu
authors.forename: James G
authors.initials: JG
authors.lastname: Wetmur
authors.forename: Madhu
authors.initials: M
authors.lastname: Kumar
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Caroline
authors.initials: C
authors.lastname: Palomeque
authors.forename: Sylvan
authors.initials: S
authors.lastname: Wallenstein
authors.forename: Jia
authors.initials: J
authors.lastname: Chen
mesh_terms: D043303:Aryldialkylphosphatase / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006239:Haplotypes; D006801:Humans; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D011247:Pregnancy; D017422:Sequence Analysis, DNA / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D043303:Aryldialkylphosphatase; C468406:PON1 protein, human