Hi!
I would like to use a slightly different approach for second strand synthesis. My problem is that I have an adapter sequence and that one somehow ligates to random sequences. I went over the protocol and I think the problem is the ligase in the standard second strand synthesis protocol by Gubler/Hofmann. Thus I would like to skip the E Coli DNA Ligase from the protocol.
The problem is that the most recommended enzymes for second strand synthesis have low processivity and therefore show Nick translation, i.e. they will fall off after 10-50bp (processivity) and then leave a nick. This one is either extended by another molecule or not. The ligase should fill these nicks. I need to find a highly processive polymerase that either degrades the RNA in the RNA:cDNA duplex or one that displaces it. Then, the reaction will run through until the end of the molecule. Is that correct?
I thought of choosing a polymerase with strong strand displacement activity and high processivity (like Phi29). Would that be a bad choice for some unforeseen reason? If has a strong 3-5 exonuclease activity but according to NEB that only applies for single stranded DNA in the absence of dNTPs. Does anybody know if that also applies for ds cDNA? Would the displaced RNA/DNA also make unwanted DNA fragments? They should not have a primer to do so, or?
Is there a highly processive enzyme (so that they can translate the nick until the 5'end of the first strand cDNA?) with 5-3 exonuclease activity commercially available?
Is there any good way to test the second strand cDNA that can be done in a lab? I tried gel analysis and that is not really indicative for total RNA.
Thanks!
Cheers,
Stephan
I would like to use a slightly different approach for second strand synthesis. My problem is that I have an adapter sequence and that one somehow ligates to random sequences. I went over the protocol and I think the problem is the ligase in the standard second strand synthesis protocol by Gubler/Hofmann. Thus I would like to skip the E Coli DNA Ligase from the protocol.
The problem is that the most recommended enzymes for second strand synthesis have low processivity and therefore show Nick translation, i.e. they will fall off after 10-50bp (processivity) and then leave a nick. This one is either extended by another molecule or not. The ligase should fill these nicks. I need to find a highly processive polymerase that either degrades the RNA in the RNA:cDNA duplex or one that displaces it. Then, the reaction will run through until the end of the molecule. Is that correct?
I thought of choosing a polymerase with strong strand displacement activity and high processivity (like Phi29). Would that be a bad choice for some unforeseen reason? If has a strong 3-5 exonuclease activity but according to NEB that only applies for single stranded DNA in the absence of dNTPs. Does anybody know if that also applies for ds cDNA? Would the displaced RNA/DNA also make unwanted DNA fragments? They should not have a primer to do so, or?
Is there a highly processive enzyme (so that they can translate the nick until the 5'end of the first strand cDNA?) with 5-3 exonuclease activity commercially available?
Is there any good way to test the second strand cDNA that can be done in a lab? I tried gel analysis and that is not really indicative for total RNA.
Thanks!
Cheers,
Stephan