BACKGROUND: Disparities in DNA template quantity across sample wells is a common occurrence in PCR experiments and a frequent cause of bogus results in research and diagnostics. High Resolution Melt (HRM) analysis, a robust genotyping technique, was shown to be highly reproducible irrespective of the reaction volume and DNA template concentration used for PCR. The aim of this study is to determine if HRM can maintain its reproducibility/reliability when varying amounts of starting DNA templates are utilized for the upstream PCR. METHODS: Serial dilutions of differentially prepared DNA templates were amplified and subjected to HRM analysis using the ABI 7500 Fast Real Time PCR machine and the ABI HRM software v2.0.6. The first serial dilution set consisted of seven 1:2 serial dilutions of amplicon-enriched DNA templates from two samples of known genotypes at exon 9 of PIK3CA, while the second serial dilution set consisted of four 1:10 dilutions of two different template types from the same sample: original FFPE DNA template and amplicon-enriched DNA template. RESULTS: HRM analysis was reproducible across two- to one thousand-fold difference in starting DNA template in the upstream PCR, so long as the starting DNA template is of ample quality and quantity to give an adequate PCR amplification. CONCLUSIONS: HRM is highly reliable in the presence of a wide variation in the amount of starting template across wells for the upstream PCR, and the factor that determines HRM reproducibility is the adequacy of the PCR amplification.
BACKGROUND: Disparities in DNA template quantity across sample wells is a common occurrence in PCR experiments and a frequent cause of bogus results in research and diagnostics. High Resolution Melt (HRM) analysis, a robust genotyping technique, was shown to be highly reproducible irrespective of the reaction volume and DNA template concentration used for PCR. The aim of this study is to determine if HRM can maintain its reproducibility/reliability when varying amounts of starting DNA templates are utilized for the upstream PCR. METHODS: Serial dilutions of differentially prepared DNA templates were amplified and subjected to HRM analysis using the ABI 7500 Fast Real Time PCR machine and the ABI HRM software v2.0.6. The first serial dilution set consisted of seven 1:2 serial dilutions of amplicon-enriched DNA templates from two samples of known genotypes at exon 9 of PIK3CA, while the second serial dilution set consisted of four 1:10 dilutions of two different template types from the same sample: original FFPE DNA template and amplicon-enriched DNA template. RESULTS: HRM analysis was reproducible across two- to one thousand-fold difference in starting DNA template in the upstream PCR, so long as the starting DNA template is of ample quality and quantity to give an adequate PCR amplification. CONCLUSIONS: HRM is highly reliable in the presence of a wide variation in the amount of starting template across wells for the upstream PCR, and the factor that determines HRM reproducibility is the adequacy of the PCR amplification.