Vu et al. BMC Genomics 2010, 11:301 http://www.biomedcentral.com/1471-2164/11/301 Page 3 of 8 Figure 1 Two-dimension BAC library pooling strategy. (a) A super pool that combines twenty five 384-well plates. (b) Two-dimension matrix pools (horizontal and vertical ones) are created from each positive super pool. The MT-PCR-HRM screen of 10 pools for each matrix will identify the positive plate (e.g. plate 23) within 2 matrix pools. (c) Then, two-dimension single pools (column pools and row pools) are created from each positive plate. The row and column pools are arranged in a way that positive BACs will appear in two row pools as well as in two column pools. Thus, from a 384 well plate containing 16 rows (A to P) and 24 columns (1 to 24), at least 8 row pools (4 different rows each) and 10 column pools (6 of 4 columns and 4 of 6 columns each) have to be generated. The PCR-HRM screen of 18 single pools (8 row pools and 10 column pools) for each positive plate will identify the positive BAC clone (e.g. row D, column 13) (d) for the marker in question. when up to three suitable markers are combined into a single MT-PCR-HRM. The practical example We use A. thaliana as an exemplar to demonstrate the usefulness of MT-PCR-HRM in combination with a mul- tidimensional BAC pooling strategy for the anchoring of markers into physical maps. The experimental design comprises of three main steps: 1) pooling of BAC freezing stocks; 2) multiplex pre- amplification of BAC pools with up to 50 primer pairs; 3) localisation of positive BAC clones by identifying the pos- itive subset of pools via selective amplification followed by HRM analysis (for each marker separately, or for up to three markers simultaneously) to confirm locus identity. 1) Pooling strategy for PCR screening of a genomic BAC library The size of large insert libraries is normally adjusted according to the haploid genome size, the mean insert size and the expected genome coverage of the library. A typical BAC library has approximately 100 Kb average insert size. It is recommended to use a BAC library with at least 3 times coverage for PCR-based screening pur- poses [5]. Such genomic BAC libraries are typically stored in microtiter plates in a 384-well format. Several pooling strategies can be applied for PCR-based screening. The number of dimensions (D) deployed in a pooling strategy defines the number subsets of pools that may contain a specific BAC clone. Screening techniques based on conventional PCR normally require 3-D or 6-D pooling systems [5,7-10] in order to ensure reliable amplification from a single BAC clone. With the more sensitive and accurate MT-PCR-HRM technique, a two- dimension pooling strategy (Figure 1) is sufficient. For studies aiming to anchor genetic markers into physical maps of large genomes, the minimum number of screen-