7.4
TITLE: RESOLUTION OF AMBIGUOUS ALLELE-PAIR TYPINGS BY HAPLOTYPE-SPECIFIC EXTRACTION (HSE)

Johannes Dapprich ¹, Harrison W Gabel ^1,2, Aruna Akkapeddi ¹, Cynthia S Turino ⁵, Sarah S Jones ⁵, Margaret Foster ³, Susan H Hsu ³, Lucille Beaudet ⁴ and Nancy B Murphy ⁵.

¹Generation Biotech, Lawrenceville, NJ; ²Dept. of Molecular Biology, Princeton University/HHMI, Princeton, NJ; ³HLA/ Molecular Genetics Laboratory, American Red Cross Blood Services, Philadelphia, PA; ⁴PerkinElmer, BioSignal, Montreal, QC, Canada and ⁵GenoVision, Inc., West Chester, PA.

Certain diploid combinations of allele pairs fail to be resolved by sequence-based typing (SBT) and sequence-specific oligonucleotide probes (SSOP). Haplotype-Specific Extraction, HSE, establishes haplotypes from individual patient's samples and permits the unambiguous typing of such combinations without knowledge of familial information.
HSE physically separates a diploid sample into its haploid components without requiring amplification: Magnetic beads are selectively attached to polymorphic sites and used to isolate the targeted fragments from a heterozygous mixture [Fig.1]. DNA separated by HSE can directly be analyzed with kits and assays already in use for HLA-typing.
Results shown are haplotype-specific separations targeting the major allele groups of the HLA-B locus; performed on genomic DNA that was isolated manually or with a robot from blood or buccal swabs. The haplo-prepared DNA was typed using commercially available kits for sequencing, sequence-specific oligonucleotide probes, fluorescent genotyping and sequence-specific priming. The purification of alleles allows the unambiguous identification of a patient's haplotype and allele pair combination [Fig.2]. (data not shown)