Vu et al. BMC Genomics 2010, 11:301 http://www.biomedcentral.com/1471-2164/11/301 Page 5 of 8 Figure 4 Multiplexing 3 genetic markers in one MT-PCR-HRM screening reaction. First order differential plots of a High Resolution Melt derived from a multiplex MT-PCR-HRM experiment (three repli- cates per treatment, green lines) performed on BAC matrix pools (5 × 384 well plates) using three independent loci as revealed on a Qiagen Rotor-Gene 6000. Positive control samples were generated using indi- vidual BAC clones containing each of the three loci (M106, red line; M34, orange line and M45, purple line). Negative control (blue line) lacked template DNA. Note that the positive control traces each occu- py a diagnostic position on the trace and that the three experimental traces yield peaks corresponding to each of these positions (green). 3) Identification of marker-containing BAC clones by high resolution melting (HRM) analysis HRM curve analysis after PCR-HRM provides a rapid, sensitive means for the BAC screening. The Rotor-Gene™ 6000 (Qiagen) with a real-time rotary analyzer prevents unwanted temperature deviation inside the thermal cycler [15,16]. Furthermore, intercalating dye improves HRM sensitivity, allows omission of the gel electrophore- sis step and enhances true positive hit rates [16]. The pos- itive BAC pools yield clear melting curves instead of a background line as obtained in the case of negative BAC pools (Figure 3). Because melting curve analysis can dif- ferentiate between divergent sequences, the option of a multiplex PCR for up to 3 loci is applicable to reduce con- sumables and labour (Figure 4). The entire MT-PCR- HRM run for a single locus (or for three loci together) can be completed within 90 minutes. The same approach is applicable to organisms of much larger genome sizes (see Table 2). For example, using the tri-plex MT-PCR-HRM option in which three loci exhib- iting non-overlapping melt profiles are combined into a single reaction, only 45 PCRs are required to screen approximately 480,000 clones of the 16 Gbp wheat genome for each of three markers represented in (an) individual BAC(s) (Table 2). Thus, this strategy poten- tially opens the possibility for high throughput-low cost screening across even very large genomes. Besides the integration of genetic and physical maps, the potential of this protocol is to support ongoing genome sequencing projects e.g., for barley http://barleygenome.org/, wheat http://www.wheatgenome.org/, potato http:// www.potatogenome.net/, swine http://piggenome.org/, cattle http://www.bovinegenome.org etc. Conclusions Here, the new MT-PCR-HRM technology has been applied to BAC matrix pools for simple, low-cost and high-throughput anchoring of genetic markers to physi- cal maps. Using a BAC library of the model plant Arabi- dopsis thaliana, we were able to show that MT-PCR- HRM can get reliable amplification of single copy targets from freezing stocks equivalent to twenty five 384-well plate super pools. A two-dimension pooling strategy can locate a target clone within a superpool by 28 reactions. The method also allows effective multiplexing to screen, in parallel, 3 genetic markers in one MT-PCR-HRM reac- tion and is particularly suited for genome characterisa- tion initiatives. Methods Primer design Primer pairs for each genetic marker (Table 3) were designed using the Primer3 program http://www/ genome.wi.mit.edu/genome_software/other/ primer3.html. The optimal features of oligo primers are: 19 - 21 nucleotides in length, ~50% GC content, average melting temperature of 57-60°C and two C/G bases at the 3'end and one C/G base at the 5'-end. Preparation of BAC pools from freezing stock of genomic BAC library We used the Arabidopsis BAC library Mi/P1[17] in this protocol as an exemplar. The library plates stored at - 80°C were thawed for 30 minutes and handled carefully in a flow cabinet to avoid contamination with clones from adjoining wells of the microtiter plate or exogenous con- tamination. After careful spin down of the plates, etha- nol-wetted (70%) paper is used to clean the lip and walls of plates. Multichannel pipettes or robotic pipetting sys- tems are used to take 5 μL of each colony for pooling into single, matrix or super pools. MT-PCR pre-amplification Pre-amplification increases the concentration of the spe- cific DNA templates in the freezing stock pools and therefore improves screening efficiency in the next step by avoiding false negative or false positive results. Modi- fied MT-PCR is performed in 20 μL containing 10 μL of