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.
Highlights
From the $Faculty of Phurmmeutical Sciences and llnstitute for Molecular and CellularBwlogy, Osaka University, Yadaoka, Suita, 565,Osaka, Japan
Mer) carrying deoxyinosineresidues (I) at positions Chemical Synthesis of Oligonucleotides-2’-Deoxyinosine and the corresponding to ambiguous nucleotides derived from aminoacidsequence have been synthesizedbythe phosphotriester method usinga polymer support
Synthesis of Deoxyoligonwkotides containing Deoxyimsine-Fig. 2 shows oligonucleotides synthesized in this study
Summary
Mer) carrying deoxyinosineresidues (I) at positions Chemical Synthesis of Oligonucleotides-2’-Deoxyinosine and the corresponding to ambiguous nucleotides derived from aminoacidsequence have been synthesizedbythe phosphotriester method usinga polymer support. The dIs have been used aqueous pyridine (50%).5’-O-Dimethoxytrityldeoxyinosine3’-0-(owhere the basein the third position of the amino acidchloropheny1)b-cyanothylphosphate was synthesized by the same codon is ambiguous. These deoxyoligonucleotidwesere procedure except that the intermediate was treated with 4 eq of 1-. Tide sequences will almost inevitably include elements of ambiguity This problem can be partly overcome by use of so-called mixed DNA probes that consist of a mixture of sequences at the ambiguous position of the codon. 1p j p 1 entire coding sequence for human gastrin cDNA was a gift from Dr
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