Niel,
I haven't played around with optimizing these reactions myself. But
just winging it... Typically, modified nucleotides have a higher Km
than the natural substrate. You might have trouble getting good
extension with just aa-dUTP. In finding the optimum ratio, you're
probably balancing the opposing goals maximizing incorporation of
aa-dUTP and poisoning the reaction. Finally, If it's deoxy it's still
DNA, albeit with a non-natural nucleotide (dUTP).
Yours,
John Thompson
Merck Research Labs
nkarrow at ovc.uoguelph.ca (Niel Karrow) wrote:
>I was wondering if anyone could please explain why one has to
>incorporate dTTP into the master mix with AA-dUTP? Is there an optimum
>ratio? It would seem to me that one could perform the RT without any
>dTTP and get a stronger flourescent signal from Cy3/Cy5 because more
>AA-dUTP was incorporated into the product. Lastly, if no dTTP was used,
>would the product not be cRNA rather than cDNA? Thanks in advance,
>Niel
