Portal de Periódicos da CAPES

[65] Use of E. coli polynucleotide phosphorylase for the synthesis of oligodeoxyribonucleotides of defined sequence

1980; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(80)65067-8

1557-7988

Shirley Gillam, Michael J. Smith,

Advanced biosensing and bioanalysis techniques

This chapter discusses the use of E. coli polynucleotide phosphorylase for the synthesis of oligodeoxyribonucleotides of defined sequence. The chapter describes a convenient isolation of polynucleotide phosphorylase from commercially available E. coli B. Oligodeoxyribonucleotides are specific mutagens for inducing defined point mutations in DNA at high efficiency. In DNA sequencing, using the terminator method, they provide specific primers for DNA polymerase and obviate the need for restriction fragments and strand separation of template DNA. Analogously, oligodeoxyribonucleotides have been used as specific primers for DNA polymerase and reverse transcriptase using RNA as template to obtain a pure radioactive probe or for sequence determinations. It is desirable to have a simple, rapid synthetic method that provides a specific, pure oligodeoxyribonucleotide because of this wide variety of applications and the large number of possible oligodeoxyribonucleotides. The procedure described in the chapter fulfills these requirements and involves the stepwise addition of deoxyribonucleotide residues to an oligodeoxyribonucleotide primer catalyzed by E. coli polynucleotide phosphorylase in the presence of NaCl and MnCl2 with the appropriate deoxyribonucleoside 5'-diphosphate as substrate.