Figure 1. Schematic illustration of the strategy adopted to isolate homeobox genes from the eye tissues.

Total RNA was extracted from micro-dissected eye components from four D3.5 eyes. Complementary DNA synthesis was primed with oligo-dT-tailed promoter sequence for the T7-RNA polymerase (dT₁₅-T7) and homeobox specific cDNAs were amplified by PCR. Degenerate primers, Hox A & B, were synthesized from the highly conserved helix 3 (H3) region of the homeobox, to serve as sense primers for PCR. The Hox B primer overlapped most of the sequence of Hox A, as it was shifted in the 3-prime direction by only three bases. The first round of amplification (PCR#1) was done using primer Hox A and T7 promoter sequence and the second round (PCR#2) was carried out with primer Hox B and the T7 promoter sequence. The amplified PCR products were then cloned and sequenced.