Bifunctional oligonucleotide probes synthesized using a novel CPG support are able to detect single base pair mutations.
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.
Nelson
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PS
Diagnostic Chemistry Division, Clontech Laboratories, Inc., Palo Alto, CA 94303.
Frye
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Liu
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D000588:Amines; D001710:Biotin; D002851:Chromatography, High Pressure Liquid; D011905:Genes, ras; D005898:Glass; D006801:Humans; D009154:Mutation; D015345:Oligonucleotide Probes; D009841:Oligonucleotides / Q000138:chemical synthesis*; D013386:Succinates
D000588:Amines; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D013386:Succinates; D001710:Biotin