A Rapid Plasmid Purification Method for Dideoxy Sequencing

The dideoxy chain-termination method of DNA sequence analysis involves the synthesis of a DNA strand from a single-stranded template (1 ). The enzymatic synthesis is initiated at the site where an oligonucleotide primer anneals to the template. Traditionally, sequencing was performed on templates produced by single-stranded phage Ml3 (2 ,3 ), or phagmid (4 ). Sequencing plasmid DNA has the advantage of eliminating the need to subclone fragments into M13, and also enables both strands to be sequenced from the same plasmid by the use of reverse primers. The most consistently satisfactory sequencing results for most purposes are probably obtained by using linear amplification techniques (cycle sequencing) either in manual procedures (5 ) or in automated sequencing machines (6 ). However, plasmid DNA that has been denatured can also serve as satisfactory template DNA for manual, nonamplified procedures (7 ). It is particularly important when preparing plasmid DNA for sequencing to give the utmost care and attention to the DNA isolation and purification techniques. Most problems that occur with plasmid sequencing are related to poor quality template.