15.9:
DNA Isolation
DNA extraction is the removal and purification of DNA from cells. First cells are lysed, broken open. Usually through a combination of physical disruption and treatment with chemicals such as detergent which dissolves the cell and nuclear membranes. The content can then float freely into the surrounding solution.
Next DNA must be separated from the other molecules present. Organic solvents such as phenol and chloroform are commonly used to separate nucleic acids from proteins based on their differing solubility.Additionally, proteinase and RNase enzymes can be used to degrade proteins and RNA leaving the DNA intact.
Salt is usually added to the sample which stabilizes the negatively charged phosphate groups in the backbone of the DNA. And after the addition of ice cold alcohol, precipitates the DNA out of the solution.
The white precipitate is collected by spinning it in a centrifuge where it settles to the bottom of the tube. After washing and re-suspending it in a buffer solution, the extracted DNA can finally be used in research or biotechnology applications.
15.9:
DNA Isolation
DNA from cells is required for many biotechnology and research applications, such as molecular cloning. To remove and purify DNA from cells, researchers use various methods of DNA extraction. While the specifics of different protocols may vary, some general concepts underlie the process of DNA extraction.
The Process
First, cells need to be lysed—broken open—to release the DNA into solution. Cells can be physically lysed using equipment such as a homogenizer, sonicator, or bead beater, which grind or otherwise apply force to break the cells open. Often, substances such as detergent are added during lysis to chemically disrupt the lipid-based cell membranes—helping release the DNA from the nucleus and cell. The spinning in a centrifuge sediments the cell debris to the bottom, and the lysate—containing cellular materials—is collected for further processing.
The DNA must then be separated from other cellular molecules, such as RNA and proteins. Therefore, enzymes such as RNase and Proteinase K are often added during or after lysis to degrade RNA and proteins, respectively. Additionally, organic solvents such as phenol and chloroform are commonly used to separate DNA from protein. Typically, the sample is vortexed with phenol-chloroform and then centrifuged to separate the aqueous and organic phases in the tube. The DNA-containing aqueous phase at the top can then be pipetted off, leaving behind the protein in the organic phase.
Salts are often added during DNA extraction to stabilize the DNA and assist in precipitating it out of solution. A standard method of DNA precipitation is to add ice-cold alcohol, such as ethanol or isopropanol, to the sample. This causes the DNA to form a white, cloudy precipitate within the liquid in the tube.
The sample is then spun in a centrifuge, causing the DNA precipitate to collect at the bottom of the tube as a pellet. The pellet is washed by removing the liquid, washing in alcohol, and spinning it again. After the final wash, the pellet is re-suspended in a buffer—creating an aqueous solution of purified DNA that is ready to be studied or used in biotechnology.