Figure 3
Sensitivity of the HUMARA assay by gel and capillary electrophoresis. To establish the sensitivity of the HUMARA assay for detection of minor alleles, different amounts of HpaII-digested (mono-allelic) and non-digested (representing bi-allelic DNA) #1290 DNA were mixed in different rations and subjected to PCR for detection of HUMARA locus. PCR products were resolved on a 3% agarose gel containing 0.5 ug/ml ethidium bromide and detected under UV light (A) or using the Applied Biosystems 3100 Genetic Analyzer (B). CTR denotes the no template control. 100, 75, 50, 25, 12.5, 6.25 and 0 indicate the percentage of bi-allelic DNA in the PCR reaction (A). Linear regression analysis calculated with Prism 6 software, shows comparison between input and detected allelic/biallelic ratios as calculated using Genescan software. (R2 > 0.98). The minor allele was reliably detectable when present at a fraction greater than or equal to 1/8 (12.5% of the input copies) (C).