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DNA polymerase with single G 3'-end overhang activity


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#1 why

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Posted 06 July 2011 - 04:27 PM

Dear all,

I came across a product webpage of GC cloning vector (http://lucigen.com/s...ART-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.

Regards,
why

#2 Trof

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Posted 13 July 2011 - 07:36 AM

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.

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#3 phage434

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Posted 13 July 2011 - 10:56 AM

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.

#4 tfitzwater

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Posted 14 July 2011 - 06:40 AM

Taq does not always add an A to the 3' end of the PCR product.
Hu, 1993 DNA and Cell Biology 12 (8) 763-770.
D. Denney, Jr. and I. Weissman 1990 Amplifications 4: 25-26.
V.L. Magnuson, D.S. Ally, S.J. Nylund, Z.E. Karanjawala, A.L. Lowe, S. Gough and F.S. Collins, 1996 Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications afor PCR-based genotyping and cloning. Nucleic Acids Res. 21:700-709.
Hite, J., Eckert, K. A., Cheng K. C., 1996. Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)nd(G-T)n microsatelite repeats. Nucleic Acid Res. 24: 2429-2434.
Costa and Weiner, 1994 NAR 22 (12) 2423.)
J. M. Brownstein, J. D. Carptena and J. R. Smith 1996 BioTechniques 20: 1004-1010.




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