Vu et al. BMC Genomics 2010, 11:301 http://www.biomedcentral.com/1471-2164/11/301 Table 3: Primer sequences used to amplify A. thaliana genetic markers. Marker ID M34 M45 M106 Forward CCAACATCGTGGGTGTTGG ATCGATCCCTACACTTCCC GGAGATGTATGTGAGTGGG Reverse AAGGAGTCATGTTTGTAGC ATTGGAGTGGCTTTATGCG AAGCATCTTAATGAAACCC Page 7 of 8 the supplied 2× Biomix (Biomix kit, Bioline), 2 μL freez- ing stock from the super pool, 5 μM of each primer (for- ward and reverse) multiplexed up to 50 markers and 4 mM MgCl2. Cycling conditions are 95°C for 10 min; then 20 cycles of 94°C for 20 s, 55°C for 30 s and 72°C for 1 min. The products are diluted in HPLC grade water to a final volume of 100 μL and stored at -20°C. To demon- strate the high multiplexing capacity of pre-amplification directly from pooled freezing stocks, 47 primer pairs (Additional file 1) from the list of 107 markers mapped to the 1.9 Mb FCA region of A. thaliana [18] were com- bined with three primer pairs shown Table 3 in one pre- amplification reaction. Subsequent application of the lat- ter 3 primer pairs for selective MT-PCR-HRM screening of the BAC library identified the BACs harbouring the 3 markers. Library screening To identify super pools (Figure 1a) that contain the posi- tive BAC with the corresponding marker, the MT-PCR- HRM reactions (10 μL) for a single primer pair in ques- tion is performed in 0.1 mL tubes containing 2 μL of the diluted pre-amplification product, 5 μL of SensiMixPlus SYBR (Quantace) and 5 μM of each forward and reverse primer. The '3-step PCR with melt' should be setup in Qiagen Rotor-Gene 6000 (Qiagen) at following condi- tions: 95°C for 10 min, followed by 35 cycles at 95°C for 20 s, 57°C for 30 s and 72°C for 50 s. To gain fluorescence for each cycling at 72°C requires the operator to choose the 'green' option. High resolution melting analysis is per- formed at ramp from 65°C to 90°C, raising by 0.3°C each step, pausing 90 s at pre-melt condition as a first step and pausing 2 s for each step thereafter. To acquire melting fluorescence, 'green' needs be chosen. In silico computer graphics reveals the live PCR run, the concentration of PCR product at the stationary stage and the subsequent melt curves. First order differential plots of the melt curves of the PCR product are created by the software provided with the Rotor-Gene™ 6000. To identify an individual plate, MT-PCR-HRM screen- ing is to be performed with freezing stocks of matrix pools as template (Figure 1b). It is possible to perform multiplex MT-PCR-HRM with up to three markers as long as distinguishable melting curves can be obtained (see Figure 4). MT-PCR-HRM components and condi- tions are as in the super pool step. After surveying posi- tive plates for every marker, positive BAC clones' ID (Figure 1d) will be identified by PCR-HRM using 2-D sin- gle plate pooling (Figure 1c) that pools across rows or col- umns to identify positive BAC clones. Markers that yield positive signals in one plate can be used for multiplex PCR. PCR-HRM components and conditions are as in the super pool step. With the list of positive BAC clones for the marker in question, the anchoring process is completed. Additional material Additional file 1 Multiplexing primers. 47 primer pairs mapped to the 1.9 Mb FCA region of A. thaliana [18] were used successfully for pre amplifi- cation to demonstrate the high multiplexing capacity. Authors' contributions GV conducted the experimental procedures. GV and MW jointly conceived the work and all authors co-wrote and approved the manuscript. Acknowledgements We thank Biohybrids International Ltd and Sumatra Biosciences for the funding of this work. Author Details 1Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Penglais, Aberystwyth, Ceredigion, SY23 3DA, UK, 2Sumatra Bioscience Pte Ltd, 8 Eu Tong Sen Street, #16-94/95 The Central, 059818, Singapore and 3BioHybrids International Ltd, 11 Penrose Avenue, Woodley, Berkshire RG5 3PA, UK Received: 23 January 2010 Accepted: 12 May 2010 Published: 12 May 2010 TB©hM2isC0ia1Gsr0etaiVncolueOmeipstceasnvl2;aAl0icl1ae0bn,ls1e1faer:3oBt0mico1l:eMhdetidspt:Cr/iebwnuwtreawdl.Lbutindo.merdthcenetraml.csomft/h1e47C1re-2a1ti6v4e/1C1o/m30m1onsAtributionLicense(htp:/creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. References 1. 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