sequencing at high temp

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Fri Jun 4 15:55:39 EST 1993


In article <thomas-030693171834 at fp1-molbio-28.uoregon.edu>
 thomas at molbio.uoregon.edu (Matthew Thomas) writes:

>I have a stech of DNA I have been trying to sequence but I am encountering
>difficulties.  I think this is due to secondary structure present in the
>single strand DNA.  I have tried sequenceing with ITP but with no luck. 
>Does anyone out there have a protocol for sequencing with Taq polymerase or
>someother high temp stable enzyme.  I think If I did the reactions at a
>high temp this might solve my problem.

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You might also consider doing your extension reactions in the presence of
single-strand DNA binding protein. If you're cycle sequencing, you might
find the following paper useful:

@ARTICLE{Chou1992,
AUTHOR = "Q. Chou",
TITLE = "Minimizing deletion mutagenesis artifact during Taq DNA
polymerase PCR by E.coli SSB",
JOURNAL = "Nucleic Acids Research",
YEAR = "1992",
VOLUME = "20",
PAGES = "4371"}

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
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