A DNA FRAGMENT WITH AN ALPHA-PHOSPHOROTHIOATE NUCLEOTIDE AT ONE END IS ASYMMETRICALLY BLOCKED FROM DIGESTION BY EXONUCLEASE-III AND CAN BE REPLICATED INVIVO

2''-Deoxyadenosine 5''-O-(1-thiotriphosphate) (dATP[.alpha.S]) was introduced into the 3'' ends of DNA restriction fragments with Escherichia coli DNA polymerase I to give phosphorothioate internucleotide linkages. Such capped 3'' ends were resistant to exonuclease III digestion. The resistance to digestion is great enough that, under these conditions, just 1 strand of a double helix is digested by exonuclease III when a cap is placed at only one end; when digestion is carried to completion, this results in production of intact single strands. When digestion with exonuclease III is limited and is followed by S1 nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced. In this way thousands of nucleotides can be selectively removed from one end of a restriction fragment. In vitro introduction of phosphorothioate linkages into one end of a linearized replicative plasmid, followed by exonuclease III and S1 nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E. coli and replicate in vivo.