| B. | Inverse PCR and sequencing of insertion site. The diagram shows the vector integrated in genomic DNA. The provirus contains two LTR sequences (big squares) in tandem and about 4 kb internal DNA. The genomic DNA/vector junctions are amplified by inverse PCR: the genomic DNA is digested with NspI, then ligated to produce DNA circles, and amplified by PCR using as primers either neoIN10 + neoIN1 (at left; called NL), or neoIN22 + neoIN21 (at right; called NR). The NspI restriction enzyme recognition site used for inverse PCR is located in the two copies of the Neo gene. The internal 4.5 kb proviral NspI fragment is too large to be amplified under our PCR reaction conditions. The sequencing primers neoES and neoLTR3 are used to sequence the junctions. By convention, we have oriented all sequences in our database as the positive strand: the insertion sites of "NL" and "NR" sequences are at the right and left end of the sequence respectively. |
