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. 2012 Mar 16;7(3):e32866. doi: 10.1371/journal.pone.0032866

Figure 9. Genotyping over a broad range of DNA amounts.

Figure 9

Melt-MAMA sensitivity to low level DNA amounts varies greatly among different assays. B. anthracis melt-MAMA targeting the A.Br.006 clade [4] accurately genotyped DNA at amount ∼19 copies. (A & B) The respective amplification plots of genomic DNA of ‘G’ and ‘A’ SNP allele templates show the amplification curves of templates titrated in ten-fold serial dilutions and in replicates of eight. The number assigned to each amplification curve (1–7) denotes the DNA amount for the starting template. (C & D) The temperature-dissociation (melt) curve derivatives for all initial template amounts are shown (numbers denote DNA amount shown). Panels C and D illustrates that genotyping accuracy is obtained across a broad range of DNA template amounts of ∼115 ng to 115 fg. Assay sensitivity to template is limited to ∼19 copies and above. An inherent characteristic of this assay is the occurrence of spurious amplification at extended cycle times (>35) in the absence of template as indicated by the NTCs. Melt-MAMAs detecting low level DNA are subject to stochastic sampling effects (B. anthracis single genome copy ∼6 fg), which is predictable using a Poisson distribution [4].