Detection of Single Nucleotide Variations in Viral RNA Populations by Primer Extension
Procedures were developed for the screening and isolation of RNA viruses that vary from the consensus population by a single point mutation and are present in low abundance. We tested these procedures using a mixture of the vaccine donor line cold-adapted (ca) B/Ann Arbor/1/66 and its wild type (wt) progenitor at a ratio of 1,000:1. A 13-mer oligodeoxynucleotide was prepared as a [32P]-radiolabeled probe which specifically hybridized to wt viral RNA (vRNA) at a region that varied between the ca and wt sequence by a single base at position 1,320 of the PA gene. This probe was able to detect as little as 88 pg of total wt vRNA blotted to nitrocellulose, while giving no positive signal with as much as 1 microgram of total ca vRNA. We were able to isolate virus containing the wt PA gene sequence from the mixed pool of ca and wt viruses by two successive rounds of amplification in embryonated eggs inoculated with a controlled number of infectious virions. During each round of amplification the abundance of the virus containing the wt PA gene sequence was increased about ten-fold. Once a relative abundance of 1:10 was reached, the virus was cloned by plaquing in Madin-Darby canine kidney cells. These procedures, which allow the isolation of specific point mutants, utilize no selective pressure conditions and are suitable for analyzing the phenotypic importance of known mutations in biologically important viruses.