Rapid and efficient methods for nucleic acid purifications are essential for many molecular biology applications. Several research groups have described methods for the purification of plasmid DNA that produce templates of sufficient quality for DNA sequence applications (1
). The introduction of capillary electrophoresis DNA sequencers, however, has put further demands on template purity when compared to automated slab gel systems. For high throughput DNA sequencing of whole mammalian genomes, purification methods must also be designed to meet the challenges of platform flexibility, increasing scalability and decreasing costs, while maintaining high quality. At the Baylor College of Medicine Human Genome Sequencing Center (BCM-HGSC), the effort required to generate a draft sequence of the Rattus norvegicus
genome in a 2 year period (13
) required a departure from the M13 DNA sequencing approach and development of a higher throughput, paired end plasmid DNA sequence strategy.
The majority of plasmid preparation protocols rely on the ‘alkaline lysis’ method, which uses a narrow pH range (12.0–12.5) to selectively denature linear, but not covalently closed circular DNA (1
). The alkaline lysate is rapidly neutralized to form an insoluble aggregate consisting of genomic DNA, protein–detergent complexes and high molecular weight RNA. In the original method the cleared lysate is then carefully recovered by centrifugation and ethanol precipitated for isolation of crude plasmid DNA. Alternative methods, such as the boiling method (2
) and microwave preparation (11
), have been used to isolate plasmid DNA; however, the alkaline lysis method represents the most robust and stable platform for further development of high throughput purification methods.
To improve the purity of plasmid DNA, several innovations have been coupled to the step of processing the cleared lysate. These methods include agarose gel electrophoresis extraction, column chromatography, cesium chloride gradient centrifugation, and selective adsorption using solid phase supports. Of these strategies, the latter has the advantages of simplicity and parallel processing via multiplexing of samples. The required centrifugation step in preparing the cleared lysates, however, significantly hinders automation by robotics. A filtration or clearing plate (9
) or selective binding conditions to carboxylated magnetic particles (12
) have been used to remove the insoluble aggregate material from the soluble plasmid DNA; however, these clearing steps have the disadvantage of increasing costs in materials and efforts, which decrease the cycle time of the method.
Glass particles, powder and beads have proven useful for purifying nucleic acids. An early technique to isolate DNA from agarose gels involved the use of the chaotropic salts sodium iodide (14
) and sodium perchlorate (15
) to facilitate binding of the DNA to common silicate glass, flint glass and borosilicate glass (glass fiber filter). Plasmid DNA initially isolated as a cleared lysate has also been purified by binding to glass fiber filter powder in the presence of sodium perchlorate (3
). Recently, Engelstein et al.
described the purification of plasmid DNA under high sodium chloride and 10% polyethylene glycol (PEG) conditions with silica particles (9
). All of these methods have the common element of high salt solutions, in which the adsorption of plasmid DNA onto the glass substrate occurs most likely by a mechanism similar to adsorption chromatography.
In the method presented in this article we utilize a direct lysis protocol, in which the lysate containing the insoluble aggregate is directly added to a 96-well filter plate containing binding solution and glass beads. Unlike the high salt methods, we have found that alcohol precipitates of plasmid DNA bind efficiently to glass substrates and can occur in the presence of the insoluble cellular aggregate lysate. The methods developed here, which allow compatibility of the direct lysis solution with plasmid DNA binding to glass beads, lends itself to a simplified high throughput method and has the flexibility for complete automation on standard robotic platforms. The cost of each plasmid DNA prepared by the glass bead method is currently ~10 cents, which affords significant advantages in high throughput sequencing of mammalian genomes. Considering genome center discounts, this represents a 4-fold saving in reagents and materials and an ~10-fold saving at market prices.