PCR & Exonuclease activity

Shahram Mori smori at nmsu.edu
Tue Jun 21 20:54:47 EST 1994


Shiao Y. Wang (sywang at whale.st.usm.edu) wrote:

: I am sequencing PCR products directly. To prepare the template, one of the
: primers is biotinylated at the 5' end. I then use strepavidin to
: isolated the single stranded DNA for sequencing. I have been getting very
: low signals lately. 

: My question: could it be that the 5' -> 3' exonuclease activity of the
: polymerase is removing the 5'-end of the product thereby removing the
: biotin?


: Shiao Wang
: University of Southern Mississippi
Shiao,
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.
Cheers,
Shahram Mori
Program in Molecular Biology
Dept of Chemistry and Biochemistry Box 3C
NMSU las Cruces NM
88003

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