Strategy for selection of candidate extending BACs based on combined use of
bioinformatics analysis and experimental molecular data. Specific primer pairs were designed on
sequenced seed BAC-end (P1 pair), on “bridge” seed BAC (P2 pair), and on potential extending BACs
(P3, P4, P5 pairs), selected from the BAC-end database. All primer pairs were tested to amplify
fragments from candidate extending BACs. Sequences of P1 and P2 fragments were aligned to the
sequenced seed BAC and to the “bridge” seed BAC, respectively, to confirm both overlapping and
direction of candidate extending BACs. Amplification of P3, P4 and P5 fragments allowed the
selection of the longest extending BAC. IL mapping through specific IL-12 lines confirmed the
position of the selected extending BAC on chromosome 12.
