Exploration of methods for sequence based HLA typing and application to patients with hair dye allergy

  • Author / Creator
    Garcia-Batres, Carlos R.
  • Determining an individual’s spectrum of human leukocyte antigens (i.e. HLA type) by sequencing their HLA genes is known as sequenced-based typing (SBT). In the first part of this thesis, non-polymerase chain reaction (PCR) methods of achieving SBT were explored. HLA-DQ cDNA sequences were cloned into a novel M13 vector so that antisense sequence was displayed, and attempts were made to specifically capture the DQ sequences using two strategies: 1) complementary oligonucleotides covalently bound to the surface of beads, and 2) complementary biotinylated oligonucleotides which were then captured using streptavadin-beads. Only the second protocol was successful, enabling enrichment of sequences about 100-fold, but the method was deemed unsuitable to achieve SBT. In the second part of this thesis an RT-PCR method to amplify the entire coding region of HLA-DPB1 transcripts was developed. By cloning and sequencing the amplified fragments, the HLA-DPB1 type could be assigned. Methods were also developed to extract total cellular RNA from human blood samples, and RNA samples from 16 hair dye-allergic patients were obtained. These were subjected to RT-PCR and HLA-DPB1 alleles assigned. The allelic distribution of the patient samples did not differ from a control population, suggesting that hair dye allergy is not associated with certain HLA-DPB1 alleles.

  • Subjects / Keywords
  • Graduation date
    Fall 2011
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.