Isolating DNA is usually easy and straightforward. However, there are sample sources that can be tricky. Some insects, for example, have a reputation of being notoriously hard to isolate DNA from, or the resulting DNA is of poor quality. In these cases, a commercial kit can be a good (though more expensive) alternative. Here I compare three commercial kits for the isolation of genomic DNA.
These genomic DNA isolation kits can be used for a variety of downstream applications such as PCR, Southern hybridization, sequencing and cloning. They can also be used with a large variety of starting materials (e.g. blood, animal and plant tissues, bacteria, yeast). The PureGene Kit from Gentra lyses cells with an anionic detergent in the presence of proteinase K and a DNA stabilizer, which limits the activity of DNases. Subsequently, RNA is removed by incubation with an RNA digesting enzyme. Other contaminants, such as proteins, are removed by salt precipitation. Finally, the genomic DNA is recovered by alcohol precipitation and dissolved in a buffered solution containing a DNA stabilizer. The Wizard® Genomic DNA Purification Kit works in a similar way but there are some slight differences: According to the manual, there is no DNA stabilizer present and though necessary, proteinase K is not included in the kit. The third kit: DNeasy Blood & Tissue Kit, works differently. It relies on the purification of samples with a silica column. Again samples are lysed in the presence of proteinase K. The lysate is loaded onto the DNeasy mini spin column which selectively binds DNA while contaminants pass through. Washing steps remove remaining contaminants and enzyme inhibitors and finally the DNA is eluted in buffer.
In our laboratory, we tested these kits to isolate DNA from various insect species like Tephritidae, Adalia bipunctata and Phyllotreta nemorum. Prior to DNA isolation, samples were ground in liquid nitrogen. The isolated DNA was used in PCR reactions and for the isolation of microsatellite markers. All three kits were successful in isolating DNA, although the yield and quality varied enormously depending on the source and amount of starting material. When isolating DNA from the flea beetle Phyllotreta nemorum, the PureGene and DNeasy kits performed more or less the same, with a yield of approximately 2-4 µg DNA from 5 individuals, compared to approximately 0,5 µg DNA from 10 individuals with the Wizard® Genomic DNA Purification Kit. A very important feature of any DNA prep kit should be the total removal of pigment, especially when isolating DNA from the Ladybird beetle Adalia bipunctata. These beetles contain a lot of pigment which can have a detrimental effect on PCR reactions. Pigment is also present in other insects where it originates mainly from the eyes. Total removal of pigment could only be achieved when using the DNeasy Kit. Apparently the silica columns of the DNeasy Kit are very successful in removing pigment. The PureGene Kit is moderately successful and the Wizard® Genomic DNA Purification Kit doesn’t seem to remove any pigment at all. The PureGene Kit is the fastest and easiest kit and the DNeasy the most laborious. Also, the DNeasy Kit uses most disposables. Although the PureGene Kit is expensive to buy, you can isolate DNA from 400 samples with it, making it the least expensive of the three kits. The DNeasy Kit is almost three times more expensive (the 96 well plate equivalent of this kit is still two times more expensive).
When isolating DNA from hundreds of samples, the best choice is to test which kit best suits your needs. (Tip: Try the trial kits most suppliers give you for free). If the DNA has to be of high quality and cost is not an important factor, you can skip this and simply choose the DNeasy Kit (or other silica based methods).
Research Associate
Laboratory for Entomology
Wageningen University