Direct sequencing of genomic DNA can close gaps in whole genome shotgun sequencing efforts and identify the DNA flanking transposon insertions. When isolating genomic DNA, it is important to make sure that the DNA isolated is high quality, since this allows for direct sequencing of the templates. Our lab has been using the Qiagen Genomic Tip G/100 (in conjunction with Qiagen Genomic Buffer Set) for this purpose and we have been happy with the results.
The G/100 columns isolate genomic DNA fragments ranging from 20 to 150 kilobase pairs (kbp) in size directly from whole blood, cultured cells, tissues, mouse tails, yeast, and both Gram-negative and Gram-positive bacteria. The G/100 column uses Qiagen's patented anion-exchange resins to bind DNA under low salt conditions. Once the DNA is bound to the column, it can be washed and eluted easily. These G/100 tips share the same technology as the Qiagen tip 100 columns supplied with the plasmid midi-kit, but are quality controlled using genomic DNA samples.
The Genomic DNA Buffer Set, which is sold separately, provides five lysis buffers to accommodate various applications, as well as the standard column buffers for equilibration, washes, and elution. These lysis buffers are specific for blood, yeast, tissue, cultured cells or bacteria. However, the Qiagen lysis protocols are not required to obtain a high quality yield of genomic DNA from the G/100 columns. For example, our laboratory isolates DNA from yeast-phase growth of the dimorphic fungus Histoplasma capsulatum using a system where the DNA is spooled, suspended in TE and treated with RNase A. Because the DNA quality after this process can be poor, based A260/A280 ratios, we pass the sample through the G/100 tips and obtain high quality genomic DNA. The purified H capsulatum DNA provides suitable template in direct sequencing reactions using the Half-Dye ready mix.
In summary, the Genomic Tip G/100 system from Qiagen yields high-quality, genomic DNA that is suitable for direct sequencing. The system is fast and easy to use and I would have no problems recommending it to anyone.
Vincent Magrini, Ph.D.
Post-Doctoral Fellow
Department of Molecular Microbiology
Washington University School of Medicine, St. Louis, MO