Direct Genomic Sequencing of Bacterial DNA: The pyruvate kinase I gene of Escherichia coli

Authors

Osamu Ohara, Harvard University
Robert L. Dorit, Harvard UniversityFollow
Walter Gilbert, Harvard University

Document Type

Article

Publication Date

9-1-1989

Publication Title

PNAS

Abstract

The genomic sequencing procedure is applied to the direct sequencing of uncharacterized regions of bacterial DNA by a "multiplex walking" approach. Samples of bulk Escherichia coli DNA are cut with various restriction enzymes, subjected to chemical sequencing degradations, run in a sequencing gel, and transferred to nylon membranes. When a labeled oligomer is hybridized to a membrane, a sequence ladder appears wherever the probe lies near a restriction cut. New probes, based on sequence that lies beyond other restriction sites, are then synthesized, and the membranes are reprobed to reveal new sequence. Repeated cycles of oligomer probe synthesis and subsequent reprobing permit rapid sequence walking along the genome. This oligomer walking technique was used to sequence the pyruvate kinase (EC 2.7.1.40) gene in E. coli without resorting to cloning or to library construction. The sequenced region was amplified by the polymerase chain reaction and subsequently transcribed and translated using both in vivo and in vitro systems, and the resultant gene product characterized to show that the gene encodes the type I isoform of pyruvate kinase.

Keywords

polymerase chain reaction, expression, multiplex oligomer walking

Volume

86

Issue

18

First Page

6883

Last Page

6887

DOI

doi.org/10.1073/pnas.86.18.6883

Comments

Archived as published.

Recommended Citation

Ohara, Osamu; Dorit, Robert L.; and Gilbert, Walter, "Direct Genomic Sequencing of Bacterial DNA: The pyruvate kinase I gene of Escherichia coli" (1989). Biological Sciences: Faculty Publications, Smith College, Northampton, MA.
https://scholarworks.smith.edu/bio_facpubs/71