Hybrid-Arrested Translation

Hybrid arrested translation (1 ) is a means of identifying recombinant DNA clones by their ability to hybridize to, and thus prevent the translation of, a specific messenger RNA in a cell-free system. In this respect the approach is similar to “hybrid selection,” also known as “hybrid release” (see Chapter 3 ), in which messenger RNAs homologous to cDNA clones are specifically trapped as RNA/DNA hybrids on nitrocelloulose filters. By washing the filters at specific temperatures and salt concentrations, RNA molecules bound nonspecifically are removed leaving only the RNA homologous to the DNA bound. This RNA can then be released by boiling the filter, precipitated with ethanol, and translated in vitro. The fundamental difference between the two approaches is that, whereas in hybrid selection translation reveals one or a few polypeptides, hybrid-arrested translation identifies a gene product by its disappearance from the pattern of translation products.