Discrimination and quantitative analysis of wild type and point mutant early region 1B genes of adenovirus using oligodeoxyribonucleotide hybridization probes.

Abstract

One of the cytocidal (cyt) mutants of adenovirus 2 (Ad2), cyt15, has been shown to have two point mutations separated by 36 bases in its early region 1B (E1B) gene that encodes the 19K (Mr, 19,000) protein. A part of the cyt15 E1B gene, including one of the point mutations, was probed with a 23-base long oligodeoxyribonucleotide (ME1B23). Another oligonucleotide probe of the same length was used for the corresponding region of the wild type (wt) E1B gene (E1B23). Over the 32P-end labeled oligonucleotide probe, a 25-fold molar excess of a competitor (1) (the unlabeled oligonucleotide of each counterpart) was added to the hybridization mixture. The E1B gene with or without a point mutation could easily be distinguished from its counterpart and quantitated in the presence of its counterpart under the hybridization conditions employed. Under the stringent wash conditions, the E1B gene of wt Ad2 could be completely discriminated from even a 100-fold molar excess of cyt15 E1B gene with the 32P-labeled E1B23 probe. The wt E1B gene could also be quantitated in the presence of a 100-fold molar excess of the mutant E1B gene.