I'm trying to sequence microsatellite markers for a plant species. Could you suggest any Taq polymerase or PCR master mix to get unambiguous sequences with sanger sequencing? Is it feasible to use Phusion high-fidelity Taq polymerase to reduce slippage during replication of repeat region? Does lowering extension temperatures help? I appreciate any suggestion ; )
Microsatellites are useful because they are highly polymorphic in the length of the repeat sequence. This means that sequencing a pcr product which is a mixture of different length repeats will look very messy after the lower number of repeats has been sequenced when you will have 2 sequences. If you are getting round this problem by cloning and sequencing clones then it will not matter much how good the enzyme is most of the clones will be the correct sequences
We used just Taq polymerase in our SSRs from the red clover. Cloned to pGEM-T vector and sequenced positive clones using vector primers.
Thanks for your comments Paul Rutland and Anastasia Shamustakimova
We will use cloning for the sequencing. Just curios about the slippage problem but Anastasia Shamustakimova made it clear, I appreciate your help
Dear Cigdem Kansu thanks for posting this ihteresting technical question on RG. You asked for literature suggestions. In this context please have a look at the following potentially useful articles which might help you in your analysis:
Microsatellite (SSR) amplification by PCR usually led to polymorphic bands: Evidence which shows replication slippage occurs in extend or nascent DNA strands
Article Microsatellite (SSR) amplification by PCR usually led to pol...
Microsatellite markers: what they mean and why they are so useful
Article Microsatellite markers: What they mean and why they are so useful
and
Rapid Development of Microsatellite Markers for Plantago ovata Forsk.: Using Next Generation Sequencing and Their Cross-Species Transferability
Article Rapid Development of Microsatellite Markers for Plantago ova...
Fortunately all three articles have been posted by the authors as public full texts on RG, so that you can freely download them as pdf files. As you can see, it always pays off to use RG directly as a valuable source of information. I hope this helps. Good luck with your research and best wishes, Frank Edelmann
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