Sensitivity and specificity of detecting minor allele fractions through High-Resolution melting analysis. G.H. Reed, Y. Wang, C.T. Wittwer. Pathology, University of Utah, Salt Lake City, UT.
   Background: Detection of minor allele fractions is important in cancer diagnosis, detection of recurrence, detection of fetal DNA in the maternal circulation, and population studies of low frequency genes. The sensitivity of sequencing is limited to an allele fraction of around 20%. Methods: Detection of allele fractions ranging from 0.25% to 50% were evaluated using a double stranded DNA dye (LCGreen Plus), real-time PCR (LightCycler) and high resolution melting analysis (LightScanner and HR-1). For PCR templates we used mixtures of genomic DNA for HFE H63D, paroxysmal nocturnal hemoglobinuria, RhD, and the 2 bp deletion in the NCF-1 gene associated with chronic granulomatous disease. In addition, three different plasmids differing in their average GC content (40, 50, 60%) were used. Homozygous fractions were mixed to achieve various allele fractions. The presence or absence of variation was assessed by a blinded investigator using melting curve shape after fluorescence normalization and temperature overlay. Two unknown melting curves were always compared to two known homozygous melting curves for assessment of sensitivity and specificity. Results: Over 2,500 different blinded calls on various targets indicate that we can identify allele fractions as small as 0.25% with a sensitivity of 95-100% and a specificity of 83-97%. Accuracy depended on the allele fraction, with lower allele fractions more difficult to call correctly. The position of the mutation, the GC content of the PCR product and the choice of instrument did not appear to affect the results. Conclusion: Allele fractions down to 0.25% can be distinguished from the wild type through high resolution melting analysis. In addition to being superior to sequencing and most existing methods in detecting minor allele fractions, our method has the added advantage that it is a simple, rapid and inexpensive closed-tube alternative, which makes sample separation unnecessary.