Genotyping over a broad range of DNA amounts.

<p>Melt-MAMA sensitivity to low level DNA amounts varies greatly among different assays. <i>B. anthracis</i> melt-MAMA targeting the A.Br.006 clade <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032866#pone.0032866-VanErt1" target="_blank">[4]</a> 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 (<i>B. anthracis</i> single genome copy ∼6 fg), which is predictable using a Poisson distribution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032866#pone.0032866-VanErt1" target="_blank">[4]</a>.