DNA polymerase with single G 3'-end overhang activity - (Jul/06/2011 )
I came across a product webpage of GC cloning vector (http://lucigen.com/store/GC-Cloning-and-Amplification-pSMART-GC-Vectors/). It stated that the non-proofreading DNA pol. (e.g., Taq, Tfl, Tth) that add a single 3´-A to PCR products have the ability to add a single 3´-G to DNA molecules as well. I would like to know under what conditions the enzymes will do so? Will this be the cause of my TA cloning failure??
Thank you very much.
Taqs make A overhangs only on some portion of product (this is usually ensured by adding final 10 min elongation step at 72 at the end of cycling) and those are degraded quicky (even frozen/thawn) so it's better to use a fresh product for TA cloning reaction. In my experience TA cloning at this conditions is very efficient, I looked at the graphs Lucigen have and I can't say I have more negative than positive colonies as they show, on the contrary, products around 400 bp are 100% efficient (10 out of 10 colonies) (Invitrogen TA kit with kanamycin resistance plasmid).
I wasn't able to find anything about G overhangs apart from telomerase function.
Maybe the enzyme adds G also in some percentage of products and G-C binding is more stable than T-A, but if you have inefficient TA cloning you may have other problems, as I wrote TA is usually very efficient.
I think Taq + dGTP (only) will add a 3' G to some fraction of the strands. Doing Pfu or Phusion PCR followed by Taq dGTP tailing would probably work quite well. But this is a lot of work, and seems only a way of avoiding someone's patent.
Taq does not always add an A to the 3' end of the PCR product.
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