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Nucleic Acids Research, 1989, Vol. 17, No. 18 7187-7194
© 1989

CHEMISTRY

Bifunctional oligonucleotide probes synthesized using a novel CPG support are able to detect single base pair mutations

Paul S. Nelson*,, Roy A. Frye1 and Edison Liu

Diagnostic Chemistry Division, Clontech Laboratories Inc., 4030 Fabian Way, Palo Alto, CA 94303 1Lineberger Cancer Research Center, University of North Carolina at Chapel Hill Chapel Hill, NC 27599 USA

*To whom correspondence should be addressed

Received March 17, 1989. Revised August 11, 1989. Accepted August 11, 1989.

A novel multifunctional controlled pore glass, MF-CPG (Fig. 1), has been synthesized and used to incorporate 3' terminal primary aliphatic amines into synthetic oligonucleotides. MF-CPG consists of a unique succinic acid linking arm which possesses both a masked primary amine for label attachment and a dimethoxytrityl protected hydroxyl for nucleotide chain elongation. Using MF-CPG, we have devised a simple and convenient technique to attach non-radioactive labels to the 3' terminus of oligonucleotides. Bifunctional probes can then be constructed by 32P labeling the 5' terminus with T4 kinase and gamma 32P-ATP Using such bifunctional oligonucleotide probes in conjunction with polymerase chain reaction (PCR) amplification, we were able to detect single base substitutions in a target segment of the human H-ras protooncogene employing either functionality. Our technique thus expands the potential applications for oligonucleotides as hybridization probes


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