Incorporation of a sequence-based HLA typing method into an immunology undergraduate laboratory course. (P4527)

Abstract

Abstract Antibody-based laboratory techniques (e.g. Western blots and ELISA) are relatively easy to implement in an immunology course and widely applicable throughout the subfields of molecular biology. Incorporating lab activities with relevance to T cell immunology is more challenging because they often require proficiency in cell culture and sterile technique. HLA typing facilitates exploration of T cell immunology, including HLA presentation and the development of a T cell response. However, clinical HLA typing kits are costly. Numerous PCR amplifications require high yields of genomic DNA, entailing the challenges of working with blood as a source for DNA in the classroom. Also, typing requires precise gel loading of many samples, which can be challenging. We transformed a sequence-based HLA typing methodology recently developed for research use with plasma samples into a protocol for an immunology course lab. Since the methodology works with the low levels of DNA recoverable from plasma, the technique is well suited for DNA extraction from buccal swabs. The HLA typing process required the following labs to complete: (1) DNA extraction and quantitation (2) primary PCR set-up (3) optional verification of amplification success and secondary PCR set-up (4) verification of nested PCR amplification and sample preparation for sequencing. Additional time was required for editing sequence data and interpreting HLA types using dbMHC.