Purifying Nucleic Acids From Samples of Extremely Low Biomass
- Created on Monday, 01 September 2008
A new method is able to circumvent the bias to which one commercial DNA extraction method falls prey with regard to the lysing of certain types of microbial cells, resulting in a truncated spectrum of microbial diversity. By prefacing the protocol with glass-bead-beating agitation (mechanically lysing a much more encompassing array of cell types and spores), the resulting microbial diversity detection is greatly enhanced.
In preliminary studies, a commercially available automated DNA extraction method is effective at delivering total DNA yield, but only the non-hardy members of the bacterial bisque were represented in clone libraries, suggesting that this method was ineffective at lysing the hardier cell types. To circumvent such a bias in cells, yet another extraction method was devised. In this technique, samples are first subjected to a stringent bead-beating step, and then are processed via standard protocols. Prior to being loaded into extraction vials, samples are placed in micro-centrifuge bead tubes containing 50 μL of commercially produced lysis solution. After inverting several times, tubes are agitated at maximum speed for two minutes. Following agitation, tubes are centrifuged at 10,000× g for one minute. At this time, the aqueous volumes are removed from the bead tubes and are loaded into extraction vials to be further processed via extraction regime.
The new method couples two independent methodologies in such as way as to yield the highest concentration of PCR-amplifiable DNA with consistent and reproducible results and with the most accurate and encompassing report of species richness.