DNA Sequencing Protocols

Volume 23 of the series Methods in Molecular Biology™ pp 9-22

M13 Cloning Vehicles

Their Contribution to DNA Sequencing
  • Joachim MessingAffiliated withWaksman Institute

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For studies in molecular biology, DNA purification has been essential, in particular for DNA sequencing, probing, and mutagenesis. The amplification of DNA in E. coli by cloning vehicles derived from M13mp or pUC made expensive physical separation techniques like ultracentrifugation unnecessary. Although today the polymerase chain reaction is a valuable alternative for the amplification of small DNA pieces (1), it cannot substitute for the construction of libraries of DNA fragments. Therefore, E. coli has served not only as a vehicle to amplify DNA, but also to separate many DNA molecules of similar length and the two DNA strands simultaneously. For this purpose, a bacteriophage like M 13 can be used. The various viral cis- and trunsacting functions are critical not only for strand separation, but also to separate the single-stranded DNA from the E. coli cell by an active transport mechanism through the intact cell wall.