Isolation of DNA from bacterial cells is usually straightforward and even amplification from crude lysates is often readily accomplished. However, some bacterial cells may pose problems and it’s often necessary to purify crude lysates as cell wall debris and polysaccharides may inhibit downstream amplification or detection. I have used Qiagen’s QIAamp DNA Mini Kit extensively on many different cell types including mammalian cells (whole blood, bone marrow, and paraffin embedded tissues) as well as various bacterial cells with favorable results. In a clinical setting, this kit was preferred since whole blood treated with a variety of anti-coagulants could be used. Additionally, for any cell type, no phenol/chloroform extraction is necessary, mitigating the hazardous waste concerns associated with phenol-contaminated byproducts. Having used many different methods to isolate DNA, I’ve found the QIAamp DNA Mini Kit to be among the least time-consuming while yielding decent amounts of pure (RNA-free) DNA. Like most kits, however, there are limitations that should be considered.
Since my current research interests are based upon the successful isolation of DNA from bacteria, the rest of this review will address these issues specifically. I’ve recently evaluated this kit using two Gram positive bacteria, Staphylococcus aureus and Bacillus anthracis. The kit contains most everything you need with the exception of lysozyme and lysostaphin (added to the lysis buffer) and 100% ethanol (added to the wash buffers). The cell lysis step is crucial for good recovery of nucleic acids and I obtained higher yields by allowing the lysis step to go for 1 hour instead of the recommended 30 minutes. Also, overloading the column negatively impacts DNA yield regardless of incubation times in lysis buffer. Lysates were then RNased (Qiagen RNase A catalogue#: 19101) and washed with the appropriate Qiagen buffer before being placed on the QIAamp Spin Column. Once bound, the DNA was washed and eluted. The entire process, including the lysis step, takes approximately 2 hours to process Gram positive cells. Our results when processing S. aureus cells using the lysostaphin protocol consistently yielded 8-10 µg of purified (RNA-free) DNA. Processing B. anthracis cells gave similar results; however, a cautionary note is warranted here. Sterility checks of DNA processed in this manner showed growth in a significant number of samples, leading us to include an additional centrifugation step on a 0.22 µm filter. Additionally, processing B. anthracis cultures that were more than 30 hours old resulted in a significant drop in DNA yield probably due to the sporulation of vegetative cells.
This kit has a high throughput counterpart (QIAamp 96 DNA Blood Kit) in 96-well format that could be adapted to work for bacterial cells. However, you will first need to array your bacterial samples in 96-well culture clusters. Cross-contamination is a potential issue here but this can be mitigated if sample volumes are kept low and a 24-channel pipettor is used to facilitate transfer. If your goal is SNP analysis from a large number of isolates or for drug discovery applications, this kit may warrant a closer look. Lastly, this kit has the most straightforward protocol and training new technicians in its use took the least amount of time.
Assistant Director
Center for Microbial Genetics and Genomics
Northern Arizona University