Haplotyping with peptide nucleic acids: a novel method for tissue typing
thesisposted on 31.01.2017, 04:24 by Murphy, Nicholas Mark
In the effort to become the first to establish the “thousand dollar genome,” a fundamental component has been outside the main focus of many geneticists: the appreciation of phase. For any genotype or allele, flanking elements can have varying degrees of influence, which limits the effectiveness of multifactorial trait/disorder mapping. Several types of laboratory means can resolve unambiguous sequences up to several hundred thousand bases in length however the longer the sequence generally means increases in time, labour and expense. This project sought to develop a means of providing a simple, robust and effective procedure to target and isolate specific DNA sequences in order to enable the unambiguous sequence. The target was decided as the human leukocyte antigen system, a cluster of genes with a well-established pattern of allelic linkage. In order to target this region, a new class of molecule, peptide nucleic acids, were bound to nominated alleles selected. The peptide nucleic acids were designed with fluorescent and biotin moieties to enable their detection and for solid phase extraction (i.e. magnetic beads/microplate) once bound to their matching allele. A probe-targetextraction methodology was used as a template for basing the protocol. To facilitate the development of the process to use on genomic DNA, two allelespecific plasmid constructs were generated to test the process under favourable conditions. Increases in the targeted allele were detected by allele specific quantification using real-time polymerase chain reaction and sequencing. The assay was adapted for use with genomic DNA, extracted from whole blood samples with a custom extraction protocol designed to maximise the molecular weight of the DNA. The assay was tested for a range of variables aimed at determining factors that influence the allelic ratio pre and post assay. Optimising the hybridisation conditions, starting amount, probe to target ratio and wash number influenced the degree of allelic separation. Further experimental work is required on yielding more consistent results in order to reliably produce haploid DNA sequences.