Use of unpurified synthetic deoxynucleotide primers for rapid dideoxynucleotide chain termination sequencing

Abstract

Synthetic oligodeoxyribonucleotides were used as primers for sequencing DNA by the dideoxy chain termination method. We report that the use of unpurified preparations of such oligonucleotides decreases the time involved in preparing the primers and yields a sequencing reaction of comparable quality to that obtained with pure preparations. It is possible to use unpurified primers if the yield per coupling step during manual or machine synthesis of oligonucleotides is greater than 98%. Also, a method for the rapid purification and desalting of synthetic DNAs is presented for cases in which the coupling is less efficient. Unpurified primers have been used to determine the sequence of a 1987-bp fragment with a single M13 recombinant clone. This DNA was first sequenced using the universal M13 primer. Subsequently, a primer homologous to the 3' end of the newly obtained sequence was synthesized and used to extend the sequence. New primers were synthesized until the sequence of the whole fragment was determined. Because only one clone is necessary, this method can be used to obtain the nucleotide sequence of large DNA fragments in a relatively short time and in an orderly fashion, simplifying analysis of the data.