Purification of DNA
Purification of DNA
Many procedures in molecular biology require an initial pure sample of DNA. These procedures include the polymerase chain reaction, sequencing, gene cloning, blotting, and DNA profiling. Purification of DNA involves removing it and other constituents from the cell, separating it from the various other cell constituents, and protecting it from degradation by cellular enzymes . Isolation procedures must also be gentle enough that the long DNA strands are not sheared by mechanical stress.
DNA can be isolated from almost any cellular source. White blood cells and cheek cells taken directly from humans are most commonly used for diagnostic purposes, but skin, hair follicles, semen, and other tissues can be used for forensic analysis. Cells grown in petri dishes or in suspension can also be used. The cells are isolated from any surrounding fluid (such as blood serum) by centrifuging them—spinning them at high speeds—and then are resuspended in a buffer solution. The buffer prevents rapid or dramatic changes in pH, which can interfere with subsequent reactions. To break open the cell membranes, a detergent is added to the buffer. Sodium dodecyl sulfate (SDS) is often used for this purpose. The detergent also helps remove proteins and lipids in the cell.
The buffer also contains ethylenediaminetetraacetic acid (EDTA), which is a chelator. Chelators are molecules that act as scavengers for metal ions in solution. This is important because DNase, an enzyme that digests DNA, is present in the cell and would destroy the long DNA strands if it was active. DNase activity requires magnesium ions, and EDTA removes them from solution, preventing DNase from cutting up the DNA. RNase is also present in the buffer at this step, to break up the RNA present in the cells.
The solution is then treated with proteinase K, a highly effective enzyme that inactivates all types of proteins. This enzyme can also be used earlier in the procedure to break apart clumped cells. Unlike many proteins, proteinase K remains active at elevated temperatures, so the solution can be heated to about 55 °C to aid protein inactivation and removal by the detergent. This step may last between two and sixteen hours.
Once the cells are broken open and the RNA, proteins, and lipids have been dissolved in the buffer, the DNA must be separated from these materials. One standard technique uses phenol to remove the proteins, leaving DNA and other water-soluble materials behind. The DNA is then extracted from the water phase using chloroform and precipitated from the chloroform using ethyl alcohol mixed with sodium acetate salt. The DNA is then removed either by spooling the long threads onto a glass rod, or by spinning it out of solution using a centrifuge. The DNA is then resuspended in buffer .
Another technique for separating the DNA from the mixture avoids the use of phenol and chloroform, which are toxic. Instead, the proteins are "salted out" by adding a concentrated salt solution and then removed by centrifuging. In another method, DNA is adsorbed onto very small glass beads, in the presence of "chaotropic" salts, which disrupt protein structure. The beads are removed (or washed in place), and the DNA is released by changing the salt concentration.
Once a pure sample of DNA has been obtained, the fragments it contains may be separated on the basis of size by gel electrophoresis . Specific sequences can be identified by Southern blotting using probes with complementary sequences, and they can then be cut out of the gel for further use, such as cloning. If the sample is small, the DNA can be amplified by the polymerase chain reaction.
A crude preparation of DNA can be made in the kitchen with simple ingredients, including baking soda (which acts as a buffer), laundry detergent, table salt, and rubbing alcohol. Meat tenderizer (an enzyme preparation) can be used to destroy proteins.
see also Blotting; Cloning Genes; DNA; DNA Profiling; Gel Electrophoresis; Polymerase Chain Reaction; Sequencing DNA.
Bloom, Mark V., Greg A. Freyer, and David A. Micklos. Laboratory DNA Science : An Introduction to Recombinant DNA Techniques and Methods of Genome Analysis. Menlo Park, CA: Benjamin Cummings, 1996.
Carlson, Shawn. "Spooling the Stuff of Life." Scientific American September (1998):74-75.
Rapley, Ralph, and John M. Walker. Biomolecular Methods. Totowa, NJ: Humana Press, 1998.