Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites

Abstract

RESTRICTION endonucleases recognise specific sequences in DNA, and these endonucleases, especially those which generate cohesive ends, have been widely used to clone DNA¹. However, many DNAs lack sequences which are recognised by endonucleases such as EcoRI, HindIII or BamHI. A general method of overcoming this problem has been described recently². This approach involves the synthesis of oligonucleotides sensitive to a specific endonuclease and the blunt end ligation of these molecules to the DNA to be cloned. In contrast, we sought a method which avoids the insertion of additional nucleotides into a DNA sequence, but depends on direct modification of DNA. If a DNA sequence differs in only one base pair from the recognition sequence of a restriction endonuclease, a particular change of this base pair will generate the proper sequence. Here we describe a way of generating restriction endonuclease cleavage sites by single base changes derived after in vitro methylation of single-stranded DNA.