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Nucleic Acids Research 2005 33(8):2615-2619; doi:10.1093/nar/gki556
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Published online 10 May 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

Molecular haplotyping by linking emulsion PCR: analysis of paraoxonase 1 haplotypes and phenotypes

James G. Wetmur1,2,*, Madhu Kumar1, Li Zhang1, Caroline Palomeque3, Sylvan Wallenstein4 and Jia Chen3

1Department of Microbiology, Mount Sinai School of Medicine New York, NY 10029, USA 2Department of Human Genetics, Mount Sinai School of Medicine New York, NY 10029, USA 3Department of Community and Preventive Medicine, Mount Sinai School of Medicine New York, NY 10029, USA 4Department of Biomathematical Sciences, Mount Sinai School of Medicine New York, NY 10029, USA

*To whom correspondence should be addressed. Tel: +1 212 241 7685; Fax: +1 212 534 1684; Email: james.wetmur{at}mssm.edu

Received January 14, 2005. Revised April 18, 2005. Accepted April 18, 2005.

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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