Overview of TVL-PCR. A. A pool of chimeric templates is generated using a ligation reaction that joins end-repaired genomic restriction fragments to TOPO vectors. Ligation is required only between one end of each genomic fragment and a vector molecule. B. A chimeric template molecule contains two priming sites (G1 and G2) within the genomic fragment and two appropriately oriented priming sites (M13F and T3, or M13R and T7) within the conjoined vector. C. In the first round of TVL-PCR, the G1 genomic primer is paired with a vector primer. In the fully nested approach an M13_ primer is used. Because the orientation of ligation is unknown, the G1 primer must be paired in separate reactions with M13F and with M13R. In the semi-nested approach, the G1 primer is paired with a T_ primer. D. In the second round of TVL-PCR, the G2 genomic primer is paired with either the T3 or the T7 primer, as appropriate. The T3 primer must be used if either the M13F or T3 primer was previously used in the first round of TVL-PCR, while the T7 primer must be used if either the M13R or T7 primer was previously used.