In genetic engineering, plasmids are used to introduce modified genes into a host organism without needing to change chromosomal DNA. This is done in order to create many copies of the DNA fragments, through the expansion of the host cells. Understanding the principle of plasmid isolation is crucial for scientists in genetic engineering, molecular biology, synthetic biology, and biotechnology. Plasmid isolation is then performed to extract and purify the plasmid DNA separately from the chromosomal DNA. Read on to learn more about the principles of plasmid isolation.
Plasmid DNA and Chromosomal DNA
One of the challenges of plasmid isolation is that most DNA extraction methods will also extract chromosomal DNA. Therefore, plasmid isolation principles rely on the differences between plasmid DNA and chromosomal DNA.
Plasmid DNA is much smaller than chromosomal DNA. Plasmid DNA typically has ten base pairs, whereas chromosomal DNA has over 4 million base pairs in most bacteria. Because plasmid DNA is so much smaller, it forms supercoils of tightly-wound double helix strands. These plasmid supercoils remain stable at pH ranges that cause chromosomal DNA strands to denature. Even if plasmids denature partially, their structure allows them to anneal quickly while chromosomal DNA remains linearly denatured.
Alkaline Lysis Principle
Alkaline lysis is the core plasmid isolation principle. Lysis is the process of breaking down a cell membrane. Alkaline refers to the pH of the solution used to lyse the cell membrane. Alkaline lysis is chemical lysis that uses high pH reagents to disrupt the proteins in the cell membrane.
The alkaline lysis plasmid isolation principle and lab technique was developed in 1979. By creating a solution that is alkaline enough to denature chromosomal DNA but not so alkaline that it denatures plasmid DNA, scientists can isolate the plasmid DNA for further study.
Alkaline Lysis Procedure
Before beginning alkaline lysis, the cells must be prepared. First, a cell sample taken from a bacterial colony is placed in a liquid solution. Next, the cells are centrifuged, and the supernatant is discarded. This is called pelleting the bacteria. At this point, the alkaline lysis reagents are added.
Typically, the reagents used for alkaline lysis are sodium hydroxide (NaOH), a strong base, and sodium dodecyl sulfate (SDS), a detergent. Chemical detergents have both hydrophobic and hydrophilic regions, so just like your dishwashing detergent, they break down fats. In this case, SDS breaks down the fatty acid portions of the cell membrane in the phospholipid bilayer, while NaOH breaks down the proteins.
Once the cells have been lysed, the solution is centrifuged again, which causes chemical reactions that neutralize the reagents. At this point, the pellet proteins are discarded because these are the proteins from the cell membranes. However, the supernatant, which contains the plasmid DNA and chromosomal DNA, is retained.
Finally, the DNA-containing supernatant is pressed through a column that filters out the chromosomal DNA but keeps the plasmid DNA. Then, elution liquid is added to the column to release the plasmid DNA from the column as it is centrifuged.
Further Steps
Additional steps can be added to the alkaline lysis procedure to wash the plasmid solution even further for specific preparations. However, the principle of plasmid isolation remains the same: use cell lysis to break cell membranes and denature chromosomal DNA while keeping plasmid DNA intact. This key technique is the basis of many genetic engineering experiments today.
Contact Hudson Robotics to discuss options on automating your plasmid isolation and DNA extraction and purification.