The Universal Primers and the Shotgun DNA Sequencing Method

For studies in molecular biology, DNA purification has been essential, in particular for DNA sequencing, probing, and mutagenesis. The amplification of DNA in Escherichia coli by cloning vehicles derived from M13mp or pUC made expensive physical separation techniques such as ultracentrifugation unnecessary. Although today the polymerase chain reaction (PCR) 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 such as M13 can be used. The various viral cis - and trans -acting 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.