DNA extraction is of key importance in biochemical research, forensic science, genomics and proteomics. There is more than one way to perform DNA extraction, for example, you may perform the method in an organic way, which involves using ethanol in the DNA precipitation step, or a non-organic way, which involves the use of lithium chloride. For the purposes of this article, we will discuss the function of ethanol and its importance in this widely used technique.
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Simple breakdown of DNA extraction
In DNA extraction, we first perform a number of steps before the inclusion of ethanol. Sequentially, we must break the cell, using a cell lysis buffer. After this, we use Ethylenediaminetetraacetic disodium salt (EDTA) to remove the membrane. When this step is complete, we use Proteinase K to remove any proteinaceous materials. At this stage, ice-cold ethanol at 95% is introduced. This is where DNA precipitation really begins. To understand how the ethanol works, we must familiarise ourselves with basic biochemical terms, like polarity and dielectric constants.
Polarity and solubility
DNA is polar, making it water-soluble. Water is also highly polar. This pertains to the "like-dissolves-like" theory, whereby solubility is based heavily on similar chemical make-up between a solute and a solvent. However, ethanol is less polar than water. This means it has a lower dielectric constant. Water has a high dielectric constant of 80.1. Ethanol has a lower constant, of 24.3. Simply put, DNA cannot dissolve in ethanol since DNA is dissimilar in structure to ethanol.
Ethanol works with Sodium Acetate
DNA's sugar-phosphate backbone is negatively-charged. For DNA precipitation to work, ethanol needs the presence of positively-charged ions (cations) such as sodium (Na+) salts. Sodium Acetate is used routinely in DNA extraction. To optimize the yield of DNA gained, it is important to consider how much Sodium Acetate is added. Excessive amounts of sodium ions equates to excess salt precipitating with the DNA. Conversely, the yield of DNA gained is poor if there is not enough salt. Ice-cold 95% ethanol plus the salts neutralizes the negative charge.
The dielectric constant is lowered
Ethanol lowers the dielectric constant and this effect intensifies the interactions between the cations and the negatively-charged nucleic acids. The non-polar nature of ethanol neutralizes the nucleic acids, which then precipitate out of solution. Additionally, ethanol induces weakened interactions between the negatively-charged phosphates and the nucleic acid polymers. This gives rise to DNA aggregation. This DNA precipitate can then be recovered after centrifuging the sample at full speed. Your DNA is now ready for further examination and subsequent storage.
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