Rapid and easy sequencing of large linear double-stranded DNA and supercoiled plasmid DNA

• Published in: Gene analysis techniques Vol. 2; no. 5; pp. 89 - 94
• Main Authors: Zagursky, Robert J, Baumeister, Kirk, Lomax, Nancy, Berman, Michael L
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.01.1985
• ISSN: 0735-0651
• DOI: 10.1016/0735-0651(85)90011-1

We have developed a rapid method for sequencing supercoiled plasmid DNA and bacteriophage λ DNA. The plasmid DNA is obtained from a 5-ml overnight culture using a rapid alkaline extraction method. The double-stranded lambda DNA is extracted from 0.75 ml of a standard liquid phage lysate using a modification of the diethylaminoethyl cellulose method. A synthetic oligonucleotide primer (12–18 bases) is annealed to the DNA, and the DNA is rapidly sequenced using the Sanger dideoxy chain-termination technique and reverse transcriptase. The advantages of these methods are 1) no cloning of the DNA is necessary, 2) one can rapidly and easily extract DNA, sequence it, and obtain the sequence in one day, and 3) both strands of the DNA can be sequenced from one molecule.