Introducing Medical Undergraduates to Germline CNV Analysis Using LILRA3: A Comparison Between Fluorescent qPCR and...

In this study, fluorescent quantitative real-time polymerase chain reaction (qPCR) and PCR-sequence specific priming (PCR-SSP) were introduced to medical undergraduates for germline copy number variation (CNV) analysis, using leukocyte immunoglobulin-like receptor A3 gene (LILRA3) as a model. This set of experiments comprises a two-session lab module requiring eight teaching hours. Using three specific primers, the wild and the deleted type of LILRA3 alleles were amplified in a single-tube PCR reaction, distinguished by the melting curve analysis (qPCR) and agarose gel electrophoresis (PCR-SSP). The CNV genotype was called for each sample using both methods, and accuracy was checked against the standard dataset. Clear Student Learning Outcomes (SLOs) were achieved, as reflected in the experimental data and the survey results. Among the eight student groups, four (B, D, E, F) excelled with both methods (accuracy rate: 90.9%-100%), qPCR proved superior for three others (C, G, H) (accuracy rate: 81.8%-90.9%), compared to PCR-SSP (0%-45.5%). Only one group (A) failed irrespective of the assay used. This laboratory exercise provides the undergraduates with an opportunity to learn about mainstream laboratory techniques for the detection of CNV, which are not commonly accessible to them, bridging the gap between theory and practice on this very important and clinically relevant topic. Upon completing this experiment module, the students showed statistically significant improvement in 10 key indexes, including the rationale understanding, acquisition of lab skills, the capability of performing fundamental genetic calculations with the genotype dataset, and personal confidence in conducting this experiment successfully (all p < 0.05).