You all know it's hard to Sanger sequence across simple sequences such as homopolymers due to slippage of DNA polymerase. I wonder if it is possible to reduce replication slippage simply by using lower temperatures during the elongation step (in both PCR and Cycle Seq) which should result in a stronger attachment of the synthesized DNA strand to the template strand. Of course one would need to adjust elongation time but normally I'm not in a hurry so that I can spend some additional minutes on that.
Has anyone ever tried this?
When running denaturing HPLC with pcr product it is clear that most of the uncommon base changes are at the ends of the amplimer so close to the primer so are probably produced at low temperatures while the cycle is warming from annealing to extension. On this basis I would say that polymerases have an optimum working temperature and at lower temperatures there are more mis incorporations and errors. There are better ways of reading through homopolymers and Laurence Stuart Dawkins-Hall
may wish to comment on this
Further on the temperature theme you could try other methods like
1 sequence in presence of 6%dmso to minimise polymer secondary structures like g quadruplex ( poly g)
2 you can sequence across a homopolymer with anchored homopolymer primmer eg t16G for a polyA tract with a c at the next base or just sequence the reverse strand
3 Modified pcr sequencing cycle where the annealing temperature is higher than the extension temperature like 94,10secs 62 for 2 mins and 55 for 2 mins
the article below may interest you
Dear Paul,
Found the answer in the file you attached: "Reduced extension temperature protocol: 30 x (96°C 5 s, 50°C 4 min)".
Thanks!
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