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AMPLIFICATION OF HAPLO-SEPARATED GENOMIC DNA BY HAPLOTYPE-SPECIFIC EXTRACTION (HSE) AND WHOLE-GENOME AMPLIFICATION (WGA).
Cynthia Turino, Johannes Dapprich, PhD, Colleen Murphy, Dylan Membrino and Nancy Murphy. West Chester PA, USA, 19382, GenoVision, Inc. and Lawrenceville NJ, USA, 08648, Generation Biotech.

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Haplotype-Specific Extraction, HSE, establishes haplotypes from individual patient's samples without knowledge of familial information by physically separating a diploid sample into its haploid components: Magnetic beads are selectively attached to polymorphic sites and used to isolate the targeted fragments of genomic DNA from a heterozygous mixture. Haplo-separated DNA is then analyzed with kits and assays in use for HLA-typing.
In cases where very little DNA is available for a particular sample or where subsequent typing is problematic for other reasons, it can be desirable to generically amplify all available DNA first. We have used the GenomiPhi™ kit (Amersham Biosciences) to amplify haplo-separated genomic DNA after HSE. The resulting amplified DNA, when typed by sequence-specific oligonucleotide probes (SSOP; Innogenetics), is still haploid with essentially no visible residual component of non-targeted alleles, and the SSOP signal generated for a haplo-separated sample is considerably stronger than for a diploid control sample.
This permits the unambiguous typing of potentially difficult diploid samples with allele pair combinations that fail to be resolved by conventional sequence-based typing (SBT) or SSOP. It should also permit the typing of haplo-separated samples with multiple polymorphisms over large linkage distances that may otherwise fail to amplify properly with locus-specific PCRs alone.