A comparison of restriction fragment length polymorphism, tetra primer amplification refractory mutation system PCR and unlabeled probe melting analysis for LTA+252 C>T SNP genotyping.

BACKGROUND

From the wide range of methods currently available for genotyping, we wished to identify a quick, reliable and affordable approach for routine use in our laboratory for LTA+252 C>T SNP screening.

METHODS

We set up and compared three genotyping methods for SNP detection: restriction fragment length polymorphism (RFLP), tetra primer amplification refractory mutation system PCR (TPAP) and unlabeled probe melting analysis (UPMA). The SNP model used was LTA+252 C>T, a cytokine gene polymorphism that has been associated with response to treatment in rheumatoid arthritis. The study was performed using 46 samples from healthy Caucasian volunteers.

RESULTS

Allele and genotype distribution was similar to that previously described in the same population. All three genotyping methods showed good reproducibility and are suitable for a medium scale throughput molecular platform. UPMA was the most cost effective, reliable and safe method since it required the shortest technician time, could be performed in a single closed tube and involved automatic data analysis.

CONCLUSION

This work is the first to compare these three genotyping techniques and provides evidence for UPMA being the method of choice for LTA+252 C>T SNP genotyping.