Enzymatic Synthesis of Deoxyribo-oligonucleotides of Defined Sequence

Abstract

THE full potential of deoxyribo-oligonucleotides in the study of replication and transcription of genetic information has not been realized because of the labour involved in the chemical synthesis of any defined sequence other than oligomers containing one type of nucleotide¹. Enzymatic synthesis, which is available for ribo-oligonucleotides², is an attractive alternative route to the use of deoxyribo-oligonucleotides. Recently, an enzyme capable of the unprimed polymerization of deoxy-ribonucleoside-5′ disphosphates has been found in E. coli W3110 po1A1³. Using a modified procedure, we have prepared a similar activity from E. coli B. This communication describes the use of the E. coli B enzyme in an approach to the stepwise synthesis of deoxyribo-oligonucleotides. The procedure allows the predominant addition of a single deoxyribonucleo-tide to the 3′-terminus of a deoxyribo-oligonucleotide. The reaction is applicable to the addition of any of the four common deoxyribonucleotides. Primers ranging from four to eight nucleotides long (other oligonucleotides have not been tested) are equally efficient acceptors. The 3′-terminus of the acceptor can be either a purine or a pyrimidine nucleotide. Unused primer can be recovered efficiently. The yield is 15–30% based on the initial amount of primer or 30–70% based on unrecovered primer.