Sequencing GC-Rich Nuclear DNA from Chlamydomonas
From Emma Berta Gutierrez, summing up several responses to a query on bionet.chlamydomonas, August 1995
egutie@ifcsun1.ifisiol.unam.mx
Here are all (or almost all) the recommendations I received from the Chlamy netters to avoid DNA compressions. Thanks a lot to everybody out there! Ember
Responses came from Dave Mitchell, Julie Knott, and Michel Ledizet
Having single-standed templates and using deaza-dGTP in the sequencing reactions gets through almost all compressions. You can use dITP but it isn’t as good as deaza.
Using TdT helps more to eliminate false stops than compression, but it REALLY cleans up the reactions, you can use it as in the USB comments article: Artifact Banding when sequencing double-stranded DNA templates by T.W. Fawcett and S.G. Bartlett. Also, constant temperature gels are recommended to eliminate compressions in GC rich DNA.Temperatures close to 55 degrees (celsius).
Using sequenase and standard TBE/urea buffer gradient gels also was another advice. Reading problems will also be present (with ss DNA) due to enzyme stops (dark bands in all four lanes), in this case Taq Polymerase won’t help.
You can use formamide in gels (30% v/v), the advice was to buy it from Fisher Biotech, the “molecular biology grade” (kept it at -20 until used). Maniatis describes how to deionize it. The gel composition was: 8M urea, 1x TBE, 30% formamide, 6% “acrylamide” (from a “30% acrylamide stock”: 28.4% acrylamide + 1.6 % bis).
We have used the two Taq kits from USB (the first version is used like the sequenase kit and the second, with a PCR protocol) with nice results.