As molecular biology techniques become more sensitive, it is increasingly important that a DNA extraction kit provides high-quality DNA. Techniques such as automated sequencing require high-purity DNA and minimal levels of contaminants, particularly inhibitory substances.
The key to an effective DNA extraction kit is the DNA-binding medium. The medium must strongly bind DNA on a large surface area to provide high yields. In addition, DNA binding media should exhibit weak binding to proteins and other molecules, so that proteins, RNA, and low molecular weight molecules can be eluted from the binding media under low-salt buffer conditions prior to elution of the DNA.
Unlike the extraction of genomic DNA, where all DNA is extracted, the isolation of plasmid DNA involves the separation of circular plasmid DNA from bacterial genomic DNA. Furthermore, the majority of the isolated plasmid DNA should remain supercoiled for increased efficiency in downstream applications such as transfection.
There are several methods for isolating plasmid DNA. A typical DNA extraction kit uses some form of alkaline lysis followed by incubation with a DNA binding matrix, washing, and then elution. An optimized DNA extraction kit for plasmids using a modified alkaline lysis method has a number of advantages including:
The scale of the plasmid DNA extraction kit used depends on the amounts of starting material and the yield of plasmid DNA that is required. Most kits come in mini, midi, and maxi prep sizes. The majority of applications use the mini kit size, mainly for screening. The midi kit is used for generating sufficient plasmid DNA for multiple manipulations or high-volume applications such as Southern blotting or pulsed field gel electrophoresis.
The exact protocol used with a DNA extraction kit for genomic DNA depends on the source. Each DNA extraction kit usually offers alternative cell lysis and wash protocols that have been optimized for common cell and tissue types.
Environmental samples often have many substances that are inhibitory to downstream applications and must be removed. For instance, soil contains high levels of humic acid, which is inhibitory to many enzymes, including Taq polymerases. Therefore, a DNA extraction kit for the isolation of bacterial DNA from soil samples must be able to effectively wash the humic acid away from the bacterial DNA.
In addition to genomic DNA, mitochondrial DNA (mtDNA) is often analyzed for forensic testing. Therefore, a DNA extraction kit designed for forensic use may be optimized to increase the yield of the small, circular mtDNA.
mtDNA has several advantages for forensics. Most cells have hundreds of copies of the mitochondrial genome and just two copies of nuclear DNA. This means that in very small samples or samples that have undergone degradation, there is a greater likelihood of getting forensically useful data.