M13 Sequencing

The sequencing of DNA has undergone rapid improvement since the introduction of the chain-termination DNA sequencing method (1 ) and the construction of convenient single-stranded DNA cloning vectors, such as the bacteriophage M13 cloning vectors and their derivatives (2 ,3 ). The chain-termination method involves the synthesis of a DNA strand by a DNA polymerase in vitro using a single-stranded DNA template. Synthesis is initiated at only one site where an oligonucleotide primer anneals to the template. The synthesis reaction is terminated by the incorporation of a nucleotide analog that will not support continued DNA elongation. The chain-terminating nucleotide analogs are the 2′, 3′-dideoxynucleotide 5′-triphosphates (ddNTPs), which lack the 3′-OH group necessary for DNA chain elongation. When proper mixtures of deoxynucleotide triphosphates (dNTPs) and one of the four ddNTPs are used, enzymecatalyzed polymerization will be terminated in a fraction of the population of chains at each site where the ddNTP is incorporated. Four separate reactions, each with a different ddNTP, give complete sequence information.