Abstract
Otago BMLSc student research project abstract.
Objectives: Secretor status of an individual is determined by the FUT2 genotype. The FUT2 genotype can be established by multiplex Polymerase Chain Reaction (PCR) - Restriction Fragment Length Polymorphism (RFLP) method using four restriction enzymes. The aim of this project was to optimise the RFLP step for clear visibility of DNA band patterns upon imaging. The subsequent aim was to establish the discrepancy rate between Lewis phenotyping and genotyping of the Secretor blood group.
Methods: Four conditions were investigated to optimise the RFLP step in FUT2 genotyping - enzyme incubation temperatures, enzyme concentrations, reaction buffers and gel electrophoresis conditions. Quantification of band intensity was performed in the Image Lab Software (BioRad) to determine whether modifications were effective. The optimised RFLP protocol was then used to genotype the secretor status of study participants recruited as part of a wider study.
Results: The optimal RFLP protocol was found to be a single temperature incubation of 37°C for 180 minutes and using a buffer recommended for one of the four restriction enzymes. Increasing the concentration of one of the four enzymes decreased the quantity of RFLP product. Gel electrophoresis ran at 100V for 90 minutes proved most effective for band separation. Of 181 participants in the wider study, 6.1% (n = 11) showed discrepancy between Lewis phenotyping and secretor status genotyping. All were of non-Caucasian ancestry.
Conclusion: The PCR-RFLP method is a valid method for secretor genotyping but requires optimisation in individual laboratories to give high performance. Discrepancy between Lewis phenotyping and secretor genotyping highlighted the unreliability of serological methods. Molecular methods are preferred for an accurate determination of secretor status.