Figure 3. The LEAP Assay.

(A) The L1 RNP minimally consists of L1 ORF1p (yellow circles) and ORF2p (blue circle) bound to the L1 RNA (multicolored line). The L1 RNP is incubated with a 5′-RACE-T₁₂VN-3′ primer and dNTPs. The L1 RT activity (green arrow) of ORF2p initiates L1 cDNA (green line) synthesis. Subsequently, the L1 cDNA is amplified using an engineered LINE-1 construct-specific primer (SENSE) and a RACE primer (red arrows), resulting in the LEAP product (double red line). (B) LEAP products can be resolved by electrophoresis and visualized by staining. LEAP products (top panel), L1 RNA present in RNPs (MMLV-RT; middle panel), and GAPDH RNA levels (GAPDH; lower panel) are shown. A standard LEAP assay includes a water control PCR reaction (H₂0); a control reaction without any RT (No RNP/RT); a reaction using RNPs from empty vector-transfected cells (pCEP4); a reaction with wild type L1 RNPs (WT LINE-1); and a reaction with RT-mutant L1 RNPs (RT mutant LINE-1). The molecular weight (MW) ladder sizes are shown in base pairs (bp). L1 ORF1p and ORF2p protein levels can also be detected by western blot analyses (not shown) (18, 28).