An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions

Abstract

Two deoxyoligonucleotide probes (23-mer and 26-mer) carrying deoxyinosine residues (I) at positions corresponding to ambiguous nucleotides derived from amino acid sequence have been synthesized by the phosphotriester method using a polymer support. The 23-mer and 26-mer corresponded to the mRNA for 8 amino acids from gastrin and 9 amino acids from cholecystokinin, respectively. The dIs have been used where the base in the third position of the amino acid codon is ambiguous. These deoxyoligonucleotides were used as probes for hybridization with colonies containing the corresponding cDNAs or genes. The hybrid formed between a gastrin clone and the 23-mer that harbors 5 dIs was dissociated at 50-55 degrees C, suggesting that deoxyinosine did not significantly effect the stabilization or destabilization of the DNA duplex. A similar result was obtained using the 26-mer that contains 5 dIs and a phage clone DNA of the cholecystokinin gene. Thus oligonucleotide probes with deoxyinosine residues at ambiguous points seem to be useful as hybridization probes for cloning genes for proteins containing amino acids with degenerate codons.