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MULTIPLEXED HAPLOSEPARATION OF GENOMIC DNA FOR HLA-A, HLA-B AND HLA-DRB1 BY HAPLOTYPE-SPECIFIC EXTRACTION (HSE).
Johannes Dapprich Ph.D. 1, Valerie McCarro 2, Cynthia Turino 2, Genaro Scavello 2 and Nancy Murphy 2. 1 Generation Biotech, Lawrenceville, NJ, USA and 2 GenoVision, West Chester, PA, USA .
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.
Here we demonstrate parallel, multiplexed HSE for three loci on the GenoM-6 / EZ-1 HSE robotic system, which processes six tubes per run in approximately one hour. The results form the basis for haplotype assembly, where several independent, multiplexed haploseparations provide DNA fragments that will allow the derivation of the molecular haplotype over extended distances.
Probes that detect specific polymorphisms within the HLA loci A, B and DRB1 were added to a single tube containing 400 ng genomic DNA of known HLA type. These probes bind to only one of the two alleles per locus. DNA fragments bound to the probes are then pulled out with magnetic beads.
The haploseparated DNA was used in three independent PCRs, where the DNA corresponding to each of these loci (A, B and DRB1) was amplified with specific primers. The amplified DNA was then HLA-typed using the InnoLiPA reverse SSOP (sequence specific oligonucleotide probe) system. The line pattern for the haploid DNA corresponds to each of the targeted alleles. The haploid DNA does not hybridize with any probe that would indicate the presence of DNA from non-targeted alleles.