Plasmid isolation and purification by electrofiltration and comparison of different direct colony sequencing methods and PCR-based sequencing methods.

Abstract

We have designed an electrophoresis system that can purify plasmid DNA from a culture without centrifugation. This system is based on electrofiltration where bacterial cell lysates are loaded in one chamber and the purified plasmid DNA is recovered in an adjacent chamber. These two chambers are separated by a membrane made of regenerated cellulose, which allows plasmid DNA to migrate to the recovery chamber while retaining most contaminants in the loading chamber. Unfortunately, even with the optimization of the parameters involved in the electrofiltration, the only DNA that can pass through the middle membrane still has some contaminants, which prevent sequencing of the plasmid. Our results have shown that a pure plasmid cannot cross a membrane with pores small enough to prevent the migration of most of the contaminants. Only a plasmid complexed with some contaminants can cross a small pore membrane. In parallel, we have compared six direct sequencing methods that do not require any plasmid purification prior to the sequencing reaction. We compared the reliability, quality of sequences, time required, and cost of these six methods. We found that the best method was that of Zhang et al. (1999). This method is fast, reliable, produces good quality sequences and is inexpensive. The performance of this method is due to the amount of ABI's ready reaction mix used, the pre-sequencing heating step to lyse the cell, the large volume of the PCR sequencing reaction and the addition of BSA.