Paraj V. Mandrekar, Zheng Ma, Steve Krueger, Cristopher Cowan, Jeff Franz, Herly Karlen and Claudio Magistrelli Promega Corporation, Madison, WI
Molecular diagnostic techniques have become increasingly informative, providing multiple determinations from an individual DNA isolate. Many of these methods, such as microarray detection, real-time PCR methods, or endpoint PCR methods rely upon high concentration DNA isolates (in excess of 100 nanogramsper microliter) to ensure success.
Most current methods used to isolate high concentration DNA require large volumes of a biological sample, such as blood, and considerable ‘hands-on’ processing time to enrich the sample as is done in the case of isolation of DNA from buffy coat samples. At the same time, there is an increasing pressure to automate DNA purification methods, where possible.
We have recently developed an automated DNA purification method that can obtain DNA concentrations in excess of 100 nanograms per microliter from submilliliter volumes of whole blood, eliminating the ‘hands on’ time required to prepare buffy coat samples. This was accomplished by combining two separate technical achievements: a novel chemistry based upon a paramagnetic cellulose particle that allows high efficiency nucleic acid capture on a small amount of particles from a sample in liquid suspension, and an LEV (Low Elution Volume) format for the Maxwell® 16 instrument that allows for elution into 50 microliter volumes. Together these two advancements allow us to purify more DNA from a sample and elute this DNA at a higher concentration than can been achieved with more traditional nucleic acid purification methodologies.
Here we will demonstrate the quantity and quality of the resulting purified DNAs using both spectrophotometric and PCR (polymerase chain reaction)-based methods.