title: Haplotype-specific extraction: a universal method to resolve ambiguous genotypes and detect new alleles - demonstrated on HLA-B.
abstract: Haplotype-specific extraction (HSE) allows the collection of individual alleles by separating diploid samples into their haploid components. The separation step is performed using magnetic beads in conjunction with allele-group-specific probes. The haplo-separated DNA samples can be directly typed with downstream applications such as sequence-specific oligonucleotide (SSO) typing, sequence-specific primer (SSP) typing, and sequence-based (SB) typing. Here we show that HSE permits the direct sequencing of an allele in its individual, separated state, including previously unknown alleles. Allele pair combinations that cannot be resolved by SSP, SSO, or generic SB typings can be unambiguously typed after the alleles are separated by HSE, which allows for new alleles to be easily detected without cloning. We show how HSE was performed to separate samples with locus-specific ambiguities in human leukocyte antigen (HLA)-B, which could not be resolved by means of generic SB typing for either sample. After haplotype-specific separation of the respective allele pairs, novel polymorphisms in the HLA-B*56 and HLA-B*44 alleles were clearly detected by SB typing.
authors.affiliation: Institute of Legal Medicine, Humboldt-University, Charite, Berlin 10115, Germany. marion.nagy@charite.de
authors.forename: M
authors.initials: M
authors.lastname: Nagy
authors.forename: P
authors.initials: P
authors.lastname: Entz
authors.forename: P
authors.initials: P
authors.lastname: Otremba
authors.forename: C
authors.initials: C
authors.lastname: Schoenemann
authors.forename: N
authors.initials: N
authors.lastname: Murphy
authors.forename: J
authors.initials: J
authors.lastname: Dapprich
mesh_terms: D000483:Alleles; D001483:Base Sequence; D005838:Genotype; D015235:HLA-B Antigens / Q000235:genetics*; D006239:Haplotypes / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D015235:HLA-B Antigens