PCR & Exonuclease activity

Shiao Y. Wang sywang at whale.st.usm.edu
Mon Jun 27 13:03:56 EST 1994


: Why don't you do direct sequencing of your PCR products using the
: Sequenase 2.0 version. It has no exonuclease activity and highly processive.
: This will take the scare out of 5'-3' exonuclease activity.

Thanks for the suggestion. I didn't think it would work. I have tried
sequencing linear dsDNA with Sequenase and it didn't work. Worked great
with circular (plasmid) DNA. Apparently, it is difficult to keep linear
DNA in a denatured state during the sequencing reaction. That's why I
purify ssDNA for sequencing. Cycle sequencing also works but not as nicely
as with ssDNA as template.

If you've had good luck sequencing with Sequenase using LINEAR dsDNA as
template, I would appreciate some pointers. Thanks again.

Shiao Wang
University of Southern Mississippi




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