Vu et al. BMC Genomics 2010, 11:301 http://www.biomedcentral.com/1471-2164/11/301 Several authors have argued that the efficiency of PCR- based screening can be greatly improved through the use of structured multidimentional BAC pooling strategies [7,8]. Compared with hybridization to high-density col- ony filters, the PCR screening of multidimentional BAC pools is less prone to the confounding effects of repetitive elements and tends to be more cumbersome when radio- labels are used [7]. Issues of error arise from both approaches, however, with the relative abundance of type 1 and type 2 errors varying according to post-hybridiza- tion wash stringency or PCR annealing temperatures respectively. Importantly, the number of independent amplifications required to determine a clone's address can be reduced by smart pooling strategies. The use of conventional PCR for BAC pool screening to anchor genetic markers to physical maps with a high throughput retains some shortcomings, most notably the common need to: i) isolate and normalise DNA from a large num- ber of clones (the whole library) to ensure that sufficient template is available from each BAC for reliable amplifi- cation of single copy targets by conventional PCR. ii) anchor and score each marker separately (multiplexing is generally difficult). iii) use agarose gel electrophoresis as crude verification (by size) of the fidelity of target amplifi- cation. This last step is simple but comparatively slow and difficult to automate. To overcome these limitations we describe a robust method for anchoring genetic markers in physical maps based on screening two-dimensional BAC pools (Figure 1) using a combination of a modified Multiplex-Tandem PCR (MT-PCR) and High Resolution Melt analysis (HRM). We demonstrate for A. thaliana that this strategy opens the possibility of direct screening of a BAC library using pooled freezing stocks for PCR with high through- put, at low cost, and a minimum risk of false positive/ negative results. Results and discussion Rationale of the method BAC library screening usually requires DNA extraction from the BAC pools [7,9] to increase template accessibil- ity sufficiently to allow reliable amplification by conven- tional PCR. We addressed the problem of low DNA template in BAC pools using a modification of Multiplex- Tandem PCR (MT-PCR) (Figure 2). The MT-PCR proto- col was originally developed to amplify rare transcripts from cDNA pools [10] but was subsequently adapted for use on genomic DNA samples [11]. The technique first comprises a truncated pre-amplification step that typi- cally consists of 15-20 cycles so that PCR amplification is arrested in the log-linear phase and template concentra- tion ratios are broadly retained. Importantly, this stage is highly amenable for multiplexing (up to 50 loci). After dilution, selective amplification is performed by conven- Page 2 of 8 tional PCR (usually 30-35 cycles) for each locus sepa- rately using either identical primers to those used for pre- amplification (as here) or new primers targeting internal sites [11]. The value of MT-PCR lies in its ability to reli- ably amplify from low copy template within mixed sam- ples. This property makes the technique highly attractive for BAC library screening for low copy targets and opens the possibility of direct screening from freezing stocks rather than from isolated BAC DNA. There remains, however, a need for a high-speed, low- cost system to verify identity of the amplicons generated by PCR (i.e. remove type 1 errors through mis-amplifica- tion) and also to distinguish PCR products from primer dimers in a gel-free environment (also creating type 1 errors). High Resolution Melt (HRM) analysis is a power- ful tool for high throughput mutation detection [12] and genotyping [13]. It therefore provides an attractive means of verifying the identity of PCR amplicons from BAC pools (Figure 3). The advantage of HRM over other geno- typing technologies for this purpose reside in its simplic- ity, high speed and low cost, the capacity to detect differences based on size and sequence, and the consis- tency of data output over a wide range of targets [14,15]. HRM analysis monitors alteration in fluorescence caused by the release of an intercalating dye from the double- stranded DNA of PCR products as they dissociate with increasing temperatures. Differences between PCR prod- ucts are revealed by melting temperature shifts represent- ing minor sequence or size variations [14]. Recent advance in fluorescence-detecting instruments (e.g. the Qiagen Rotor-Gene 6000) allows HRM to be performed immediately following amplification and to be completed within 15 min. Careful selection of amplicons that have non-overlapping melt profiles also opens up the possibil- ity of multiplexing HRM analysis (Figure 4). Multiplex pre-amplification and HRM analysis make BAC library screening easy and minimize the risk of false positive or false negative results that can appear when pooled freez- ing stocks are directly used for PCR. Our MT-PCR-HRM-based protocol describes a rapid and robust way to identify low abundance positive clones within large insert libraries. A positive superpool is iden- tified by N/25 MT-PCR-HRM reactions where N is the number of 384-well plates of the BAC library and a super pool contained 25 384-well plates (Table 1). A positive plate will be defined by two positive matrix pools after 10 MT-PCR-HRM reactions. The positive clone of a given plate is identified by the same 2D pooling strategy from four positive single pools (2 row and 2 column pools, Fig- ure 1c) after 18 PCR-HRM reactions (Figure 1 and Table 2). In total N/25 + 28 MT-PCR-HRM reactions are needed for each marker when tested separately (Table 1). The number of MT-PCR-HRM reactions can be reduced