Melt Analysis of Mismatch Amplification Mutation Assays (Melt-MAMA): A Functional Study of a Cost-Effective SNP Genotyping Assay in Bacterial Models
Figure 10
Competition between specific and non-specific amplification at low level DNA amounts.
The B. anthracis Melt-MAMA targeting the A.Br.006 clade [4] stochastically amplified allele-specific product and non-specific spurious products at amounts of less than ∼19 copies. (A & B) The respective amplification plots of genomic DNA of ‘G’ allele and ‘A’ SNP allele templates show the amplification curves of templates at 1.15 ng and at two low level ten-fold dilution series (∼19 copies and near a single copy) in replicates of eight. The number assigned to each amplification curve (3, 7–9) denotes the DNA amount for the starting template. (C & D) The temperature-dissociation (melt) curves derivatives for the 1.15 ng and lowest template amounts are shown. This panel illustrates that assay sensitivity to template is limited to ∼19 copies and above. Below this template amount, spurious amplification is possible and difficult to differentiate from template-specific amplification.