A few hundred bacterial cells obtained by touching a bacterial colony with a sterile toothpick can be used directly in a polymerase chain reaction (PCR) amplification procedure to identify and orient a plasmid insert (1,2). By combining this procedure with one in which asymmetrically amplified DNA is used for sequencing (ref. 3 and Fig. 3), we have demonstrated that DNA amplified from a bacterial colony can be sequenced directly by the dideoxy chain-termination method to yield results as good as those obtained when purified template DNA is used for amplification (ref.4 and Fig. 2). By end-labeling the primer that is used in limiting amounts during the amplification step and using it for sequencing, an entire insert of 300 nucleotides or less can be sequenced in one step. Inserts of larger size can be sequenced by using labeled primers that bind within the amplified single-stranded DNA sequence. The procedure is rapid and enables one to obtain sequences from as many as 20 clones in a single day.
Available at: http://works.bepress.com/david_brian/29/