Cloning in Single-Stranded Bacteriophage As an Aid to Rapid DNA Sequencing

An approach to DNA sequencing using chain-terminating inhibitors (Sanger et al., 1977) combined with cloning of small fragments of DNA in a single-stranded DNA bacteriophage is described. Random fragments from restriction enzyme digestion of the DNA are inserted into the EcoRI site of the modified bacteriophage M13mp2 (Gronenborn & Messing, 1978) using a linker oligonucleotide. Individual recombinant plaques are collected, 1-ml cultures grown, and the DNA isolated. A "flankingprimer" from the vector is used to determine a nucleotide sequence in each inserted DNA fragment by the chain-terminating method. This is a relatively rapid and simple method of accumulating sequence data. The 2771-nucleotide sequence of the largest MboI restriction enzyme fragment from human mitochondrial DNA was determined by this method.