Genomic DNA (gDNA) is chromosomal DNA that holds the complete set of genetic data for an organism – unlike plasmids, were the DNA is extrachromosomal. Sizes and molecular weights of various gDNAs can vary from organism to organism.
The highest DNA yield and quality is achieved by purifying gDNA from freshly harvested tissues and cells. If circumstances make it difficult to process the sample directly, then it is important that you make sure to:
- Store at conditions that preserve DNA integrity, depending on the starting material used
- Use stabilization reagents
- Limit repetitive freezing and thawing of your frozen samples
Enzymatic digestion using different enzymes such as Proteinase K or lysozyme are useful to lyse soft tissue, cell culture or blood and bacterial cells respectively. For tougher samples, mechanical disruption aids in successfully preforming the lysis step and may be performed by using either a mortar and pestle with liquid nitrogen, a handheld rotor-stator (e.g., TissueRuptor) or a bead mill (e.g., TissueLyser LT or TissueLyser II). Fixed samples require: 1) an initial deparaffinization pretreatment step to remove the wax before continuing with Proteinase K to lyse the sample, and 2) a heat treatment step to reverse formalin crosslinking.
4 common DNA extraction technologies, each having their advantages and disadvantages:
|DNA extraction technologies:||Advantages:||Disadvantages:|
|Phenol/chloroform||Efficient lysis, high yields||Toxic agents, variable results, carryover|
|Anion-exchange||Delivers higher molecular weight DNA (~100 kb)||Slow|
|Silica membrane technology||Delivers highly pure DNA, easy to use, fast, inexpensive||Difficult to automate|
|Magnetic particle technology||Manual use delivers higher molecular weight DNA (~200 kb), fast, inexpensive||Bead carryover|
A common pitfall in using commercialized kits for DNA extraction is overloading the column. When the column clogs up, the obtained yield is low. Therefore, always be cautious not to overload it.
To check on the quality and concentration of gDNA, you can use UV measurements to look at different wavelengths and ratios. An alternative method would be to use fluorophores. Whichever method you use, always be aware of using these measurements as general guides rather than absolute criteria.
For more tips and suggestions on gDNA isolation, check out the webinar slide deck on SlideShare!