Second-strand synthesis - why use ligase? - DNA polI - mediated SSS (May/20/2008 )
I am doing second-strand synthesis of cDNA. Most protocols say to use RNaseH to nick the RNA strand, Klenow or DNA pol I to synthesize the second strand from the RNA primer, and E. coli DNA ligase to join the second strand fragments together.
I don't have E.coli DNA ligase but I am wondering if I need it if I use DNA polymerase I (as opposed to Klenow fragment) because doesn't it have a 5'->3' exonuclease / nick-translation activity anyway?
Isn't that a simple PCR with both F and R primers ?
I don't see why you bother with ligase, klenow etc etc... If you do a simple PCR, the denaturation step wil get rid of your RNA... The way you are presenting it makes me think of DNA replication in eukaryotes, with the Okazaki fragment... you know...