A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes.

P.M Howley,M.A Israel,M.F Law,M.A Martin

doi:10.1016/s0021-9258(17)30093-5

Abstract

A new approach for evaluating homologous sequences among related DNAs is presented. Conventional filter hybridization techniques are employed at 35 degrees C in a range of formamide concentrations in order to perform annealings at effective temperatures as low as Tm -50 degrees C which permits the detection of regions of homology with as much as 33% base mismatch. Under such nonstringent conditions, high levels of specific annealing can be obtained at plateau levels. In combination with the Southern "blotting" technique (1975), this approach can be used to perform biochemical heteroduplex melting experiments. The homology among the genomes of the murine polyoma virus (Py), the simian virus 40 (SV40), and the human papovavirus BK was evaluated using this new methodology.

Highlights

In order to demonstrate the approach described in this paper, we have evaluated the sequence homology among simian virus 40 (SV40), human papovavirus BK (BK), and polyoma virus (Py)
DNA in solution (DNA,), and its hybridization to the DNA immobilized on the filter (DNAf) (Flavell et a&, 1974)
We have presented a new technique for evaluating homology among related DNAs, which is based on current nitrocellulose filter hybridization methodology

Summary

EVALUATION

There are a number of situations, (i.e. the evaluation of the evolutionary relationship and sequence divergence of related DNAs or the study of genes coding for similar proteins) where one must be able to detect homologous regions containing greater base mismatch In this manuscript we present a method for rapidly evaluating and mapping related sequences occurring in different DNAs. Conventional hybridization techniques were adapted to a series of less stringent conditions by carrying out hybridizations to immobilized DNA at 35’C using a range of formamide concentrations. Using the same approach as Ferguson and Davis, Newell and co-workers demonstrated extensive homology throughout the early regions of the genomes of BK and SV40 at T, - 35°C (Newell et al, 1978) providing further evidence that evaluation of DNA homology under stringent hybridization conditions can be misleading and fail to detect clearly related regions of DNA.

PROCEDURES

RESULTS

DISCUSSION