Researchers are often interested in analyzing a certain sequence of DNA. This sequence might be a region that encodes a protein or it may be part of the regulatory area of a gene. In order to analyze the sequence it is often necessary to obtain a large amount of the DNA. One of the ways this is done is by inserting the sequence into a plasmid, a small, circular unit of double-stranded DNA. This plasmid is then placed into bacteria such as E. coli by a process called transformation. The bacteria act as a host and creates large amounts of the plasmid. After critical mass has been reached the plasmid DNA is isolated from the bacteria. The trick is to not only isolate the plasmid DNA from bacterial proteins and lipids, but also from the genomic DNA of the bacteria. This process can be divided into two steps.

The first step is to break open the bacterial cells, a process called lysis, and separate the plasmid and genomic DNA. Two different conditions are used to do this:

  1. Alkaline Lysis:
    This is the most commonly used condition. First, the bacteria are lysed in a solution that contains sodium dodecyl sulfate (SDS), a detergent that denatures proteins and disrupts the cell membrane. Next, sodium hydroxide (NaOH) is added, which makes the solution very alkaline (hence the name of the method). This causes the plasmid and genomic DNA to denature into single strands. The basic solution is then brought to a neutral pH by adding potassium acetate (KAc). The genomic DNA is very large and is unable to reanneal back into double-strands. It therefore cannot remain in solution and forms a precipitate. However, because the plasmid DNA is much smaller than the genomic DNA it is able to reanneal when the KAc is added, and therefore it remains dissolved in the solution. KAc also precipitates the SDS and most bacterial proteins and lipids that are bound to it. The contents are spun in a centrifuge and the supernatant (containing the plasmid) is removed from the precipitate (containing genomic DNA). Simple kits can be purchased that isolate plasmids using this method.
  2. Boiling Lysis:
    This method is similar to alkaline lysis, except here hot temperatures are used to denature the plasmid and genomic DNA. The bacteria are placed in a boiling water bath which lyses the cells and denatures the DNA. The solution is cooled, causing the plasmid DNA to reanneal and remain in solution while the genomic DNA precipitates. The bacteria are spun in a centrifuge and the supernatant containing the plasmid DNA is removed. This technique appears to be older and used much less frequently than the alkaline lysis technique.

These two techniques work well at isolating plasmids from varying quantities of bacteria. If plasmids are isolated from a small amount of bacteria (about 5 mls) then the process is called a "miniprep". If larger amounts of bacteria (from 500 ml to one liter) are used it's referred to as a "maxiprep".

After finishing the first step the plasmid DNA is in a solution with small fragments of genomic DNA and bacterial proteins, lipids, and RNA. In the second step the plasmid DNA must be further purified from these remaining contaminants. There are several different ways to do this. The easiest way is to first remove the RNA by treating the solution with RNase, an enzyme that only digests RNA. Next the solution is mixed with phenol and chloroform, which helps to isolate the DNA from remaining proteins and lipids. The DNA is then purified with an ethanol precipitation. This method is very fast and simple.

Another way to purify the plasmid DNA is through a technique known as cesium chloride gradient centrifugation. Two compounds, cesium chloride and ethidium bromide, are added to the solution. Ethidium bromide is an intercalator, meaning it inserts itself between the base pairs of DNA. Linear DNA pieces like the genomic DNA fragments bind more ethidium bromide than the circular plasmid DNA. The solution is placed into a tube that is spun extremely fast (roughly 50,000 revolutions per minute) in an ultracentrifuge for about a day. During this time the cesium chloride forms a gradient of lower density at the top of the tube and higher density at the bottom. The genomic and plasmid DNA form tight bands in this gradient. Since the plasmid DNA binds less ethidium bromide it is more dense and is located lower in the tube than the genomic DNA. RNA forms a separate band at the bottom of the tube. These three bands can be visualized by UV light. A needle is used to puncture the tube to collect only the plasmid DNA band. This process results in extremely pure plasmid DNA, but the method is expensive and time consuming. Ethidium bromide is also a mutagen, so great care needs to be taken to avoid exposure.

Finally, plasmid DNA can also be purified by running the solution over a column. Anion-exchange chromatography can be used to isolate only the negatively charged plasmid DNA. Size-exclusion chromatography can also be used to separate the large plasmids from the smaller proteins, RNA, and genomic DNA.

After purification researchers are generally left with anywhere from a microgram to a milligram of plasmid DNA. This DNA may then be sequenced, inserted into eukaryotic cells, or used for any number of other techniques to get a better understanding of the DNA sequence.



Sources:

  • Current Protocols in Molecular Biology, Volume 1, 1998
  • http://uregine.ca/~ngdann/Bioc422/PROJ2.htm

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