Keeping Pace Downstream
Many high-throughput screening regimes use blood, tissue, hair, or mucosal samples for PCR, trait, and viral diagnostic analysis. While many of these methods are highly effective at producing good-quality DNA, relatively few are easily automated, and many of the steps expose the sample to potential contamination by the technician or extraneous DNA.
prepGEM is effective for extracting DNA from blood, saliva, tissue, follicle, milk, mucosal, and bacterial swabs. The proteinase works well when DNA yields are high and dilutions are required prior to PCR amplification (as is the case with fresh blood). However, a real benefit of the method is the high-throughput automation and ability to scale down to small volumes. The simplicity and closed-tube nature of the method maximizes yield and the compatibility of the extraction buffer with PCR means that the extract can go directly into the amplification reaction.
The extraction procedure involves heating a buffered suspension of sample to 75°C for 15 min in the presence of buffer and enzyme (Figure 1). Buffer and/or sample volume can be easily altered to control yield while maintaining enzyme concentration. As little as 5-µL extraction volume can be used for just a few cells, or as much as 850 µL for a whole buccal swab has been successfully demonstrated. A subsequent incubation for 5–15 minutes at 95°C kills the enzyme, and the extract is available for immediate PCR.
The main advantage of using a proteinase that is optimally active at high temperatures is that DNA is released from the cellular material at temperatures where contaminating nucleases are inactive. Nucleases are therefore rapidly hydrolyzed by the proteinase before any degradation of the DNA occurs.