Lambda Exonuclease and Sequencing PCR Products

Robin Beech robin_beech at
Fri Jan 19 13:45:24 EST 1996

I am interested in sequencing a PCR fragment using the method of Higuchi
and Ochman (1989). The fragment is 500bp long and sequencing single
stranded template produced by asymetric PCR works, but not all that well.
The Higuchi method relies on the fact that lambda exonuclease will only
degrade DNA if a 5' phosphate is present. If one primer is phosphorylated
prior to regular PCR then treatment of the product with lambda exonuclease
should remove that strand and leave a single stranded template for

Unfortunately whenever I treat my PCR products with the exonuclease, even
without prior phosphorylation, they disappear. DNAgency who made the
primers say there is no way the primers could already have a phosphate.
Treating DdeI digested pGEM-3z with the exonuclease degrades the DNA.
Treating the plasmid digest with alkaline phosphatase protects it, so the
requirement for the 5' phosphate is there.

Does anyone have any ideas as to why an unphosphorylated PCR product would
be degraded this way, and what I can try to remedy the situation?

Robin Beech
robin_beech at
Institute of Parasitology
McGill University

Higuchi and Ochman (1989) Production of single-stranded DNA templates by
exonuclease digestion following the polymerase chain reaction. Nucleic
Acids Research 17: 5865

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