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Nucleic Acids Research, 1993, Vol. 21, No. 23 5332-5336
© 1993

METHODS

Single base pair mutation analysis by PNA directed PCR clamping

Henrik Ørum*, Peter E. Nielsen1,*, Michael Egholm2, Rolf H. Berg3, Ole Buchardt3 and Christopher Stanley*

PNA Diagnostics A/;S Lersø Park Alle 42, DK-2100 Copenhagen ø 1Research Center for Medical Biotechnology, Department of Biochemistry B The Panum Institute, Blegdamsvej 3C, DK-2200 Copenhagen N 2Department of Organic Chemistry, The H.C.sted Institute Universitetsparken 5, DK 2100 Copenhagen Ø 3Polymer Group, Materials Department, Risp National Laboratory DK-4000 Roskilde, Denmark

*To whom correspondence should be addressed

Received September 16, 1993. Revised October 19, 1993. Accepted October 19, 1993.

A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity than the corresponding deoxyribooligonucleotides and that they cannot function as primers for DNA polymerases. We show that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA is targeted against one of the PCR primer sites. Furthermore, we demonstrate that this blockage allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers.


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