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.
Vu et al. BMC Genomics 2010 11:301 doi:10.1186/1471-2164-11-301