Journal
NUCLEIC ACIDS RESEARCH
Volume 33, Issue 8, Pages 2615-2619Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gki556
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Funding
- NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES [R21ES011643, P42ES007384, P01ES009584] Funding Source: NIH RePORTER
- NIEHS NIH HHS [R21 ES011643, P01 ES09584, P42 ES007384, P01 ES009584, P42 ES07384] Funding Source: Medline
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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.
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