Vu et al. BMC Genomics 2010, 11:301 http://www.biomedcentral.com/1471-2164/11/301 Page 4 of 8 Figure 2 The need for pre-amplification: MT-PCR-HRM using freezing stocks of a super pool (25 × 384 well plates). A first order differential plot showing failed amplification by direct PCR (35 cycles, red line) and in the negative water control (blue line) but a strong sig- nal after preamplification applying MT-PCR-HRM (green line) for STS marker M45. ing reactions is decisive in setting labour and consum- ables costs. The pooling strategy proposed here allows screening of millions of clones with as few as the number of superpools plus 28 MT-PCR-HRM reactions (Figure 1). Based on our experience, each super pool should not comprise more than twenty five 384-well plates. Using 5 μL per BAC clone, a super pool contains in total 5 × 25 × 384 μL (= 48 mL). In our illustrative example, an A. thali- ana library [16] was used that contained twenty four 384- well plates. To evaluate whether the enhanced sensitivity of this method has utility for organisms with much larger genomes, we simulated 'rare single locus' hits in a sub- stantially larger library by creating a superpool contain- ing 5 μL of a single positive BAC clone and 3200 ([25 × 384]/3) negative BAC clones of 15 μL each. We were able to exploit this approach to estimate the capability of the technique to detect single locus hits for genomes of much larger size (Table 2). According to the proposed pooling strategy, the required number of super pools for a typical 3× coverage BAC library with a 100 Kb average insert size is estimated for several important genomes (Table 2). For genomes larger than 1500 Mb, the number of PCRs required to anchor one genetic marker is approximately equivalent. The number of plates per superpool can be varied in a certain frame (i.e., less than 25) according to the genome size of the organism under study. Figure 3 Screen of Matrix pools by MT-PCR-HRM. First order differ- ential plots of a High Resolution Melt derived from a MT-PCR-HRM ex- periment for STS marker M106 (three replicates per treatment) performed on BAC matrix pools (5 × 384 well plates) containing a sin- gle target BAC (green line) or on the same matrix pool but lacking the target (pink line). All HRM analyses performed on a Qiagen Rotor-Gene 6000. 2) MT-PCR pre-amplification Applying a suitable pooling strategy, the number of PCR amplifications required to identify the positive BAC clones can be reduced significantly. However, because of a low template concentration for STS sites and further dilution by E. coli DNA, conventional PCR-based screen- ing may over-dilute single locus targets to such an extent that amplifications often fail. DNA extraction from the BACs is required for better template accessibility [7,9]. Instead, we introduced a multiplex pre-amplification step on super BAC pools (each containing twenty five 384- well plates) directly from freezing stocks, applying up to 50 primer pairs simultaneously. Before pre-amplification, it has to be confirmed that each primer pair applied works individually on total genomic DNA by PCR-HRM. The pre-amplification increases the sensitivity and specificity of MT-PCR-HRM on pooled templates (Figure 2). Without pre-amplification a higher cycle number is generally required for conventional PCR (40 to >45 cycles), yielding an excessively high background to prod- uct ratio (Figure 2).