Fig. 2.

ADP-AlF–dependent spRNA synthesis requires the nontemplate strand −12 to +3 sequence. Denaturing gels demonstrating that truncated templates give rise to a slower-migrating species: product Y (compared with the RNA marker, lane M; USB). Notably, the spRNA product (with a 5′-OH) migrated slower than a 10-base RNA marker (5′-³²P-labeled), but cold kinasing the 5′ end (Materials and Methods) resulted in the expected spRNA migration (lane 2, *spRNA). A further control illustrates that the spRNA product is sensitive to RNase TI but not DNase I; however, product Y was sensitive to DNase I and was subsequently identified as nonspecific incorporation of [α-³²P]GTP on one DNA strand. The truncated probes used in this study with the consensus GC and regions of mismatch in bold are indicated.