Q - Which of your modified nucleoside triphosphates can be incorporated into DNA molecules through PCR?
A – Aminoallyl-dNTPs, biotin-AA-dNTPs, 2-amino-dATP, 7-deaza-dGTP, and 7-deaza-dATP, 5-Methyl-dCTP, 5-Iodo-dUTP, 5-Bromo-dUTP, 5-Fluoro-dUTP, N4-methyl-dCTP, 5-propynyl-dUTP and 5-propynyl-dCTP, 2-thio-dTTP, 4-thio-dTTP and alpha-thio-dNTPs can be incorporated through PCR. As not all of these analogs will incorporate with similar efficiencies in PCR, some optimization may be needed. We recommend performing some initial PCR experiments using natural:modified dNTPs in ratios such as 1:0, 3:1, 1:1, 1:3 and 0:1 to identify the best conditions for modified nucleotide incorporation with robust amplicon yield.
In addition, 2'OMe-dATP and 2'Fluoro-dCTP can also be incorporated using a modified taq polymerase called Therminator III from NEB. Just ask Mimi. It needs to be optimized, but does work. 2'OMe-dA does not work as well though.
(hint, just click on the image above to see the whole thing)
Methods: A 10cm 10% Native Acrylamide self poured into ready gel housing with long wells was used. 10ul sample + 2ul 5X BioRad dye (Cat # 161-0767) was loaded onto gel. 25 cycles PCR with 45 second steps. 0.1 pmol target.
Observations: 10% Native Acrylamide Gel is easy to pour and takes only 25 minutes to run on these rigs. This gel ran for about 25 minutes at 250V and the darker bromophenyl blue was 1cm from the bottom. The 70bp amplified products midway through the gel with all polymerases tested.
Results: A 70bp product was detectable on the gel for all polymerases with normal nucleotides. However, there were also artifact bands detectable suggesting mispriming or inappropriate cycling conditions. Vent seemed to work the best with the cleanest darkest band for this template using 2’Fluoro dC. Therminator produced artifacts with 2’F dC also. Therminator also produced some smaller termination products with 2’Ome dA seen in lane 3 and 4. Taq polymerase could not incorporate any unnatural nucleotides and also did not show the characteristic banding pattern with natural nucleotides.
Conclusions: The thermo taqs (Therminator, Vent and Vent (-exo) produced banding artifacts with the normal set of nucleotideshese could largely be reduced with modified nucleotide 2’Fluoro-dC. . Therminator, Vent and Vent (-exo) were able to incorporate 2-F dC. None of the polymerases were able to adequately incorporate 2’Ome dA to a sufficient level detectable on this gel. 2’O-Me dA was not a good nucleotide for incorporation enzymatically. For future experiments, use 2’F dC with Vent for incorporation.
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