DNA & RNA Extraction Function & Applications
DNA Extraction and RNA extraction are key to acquiring deoxyribonucleic acid / ribonucleic acid from cell or virus samples. It is important to know that purification, extraction, and isolation interchangeable terms but can refer to slightly different processes. Purification usually relates to eliminating contaminants, while isolation is removing as much of the target as physically possible. Extraction can combine both of these terms or be similar to isolation.
DNA extraction is vital to the downstream processes of PCR, sequencing, and genotyping, while RNA extraction is key for gene expression studies, gene knockdown, gene silencing processes and transcriptome analysis. However, isolating RNA is a meticulous process due to the presence of RNases within cells. These enzymes require inhibiting as they can rapidly degrade RNA and reduce the overall yield.
Both DNA & RNA extraction are vital first steps for preparing the final sample.
DNA Purification, which commonly uses magnetic beads or silica columns, requires these steps:
• Cell lysis via the use of:
* Chaotropic Salts – make it possible for DNA to bind to solid phase (e.g. silica membrane)
* Detergents – able to degrade proteins and cellular structures, including the nuclear envelope
* Enzymes – such as proteinase K and RNases, which break down proteins and RNA respectively
• Removal of contaminants (e.g. carbohydrates) through centrifugation and pipetting, specific methods include:
* Ethanol Precipitation – DNA is insoluble in alcohol
* Phenol-Chloroform Extraction – Phenol removes proteins in the aqueous layer
* Mini-column / Spin Column Purification – DNA will bind to silica under the correct conditions
Check out our article about the Coral Reefs and DNA extraction tips for working with this sample type.
RNA Purification, in general, runs as follows:
Guanidine thiocyanate and a reducing agent disrupt the cells
Vigorous shaking or vortexing break any disulfide bonds as well as inactivating any contaminants
Phenol and chloroform-isoamyl, followed by centrifugation, separate RNA in the cell
Ethanol washing (75%) removes impurities from the precipitate.
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