DNA Extraction

Any biological fluid or tissue that contains nucleated cells can be used as a source of DNA. A nucleated cell is any cell that has a nucleus. In blood the white blood cells have nuclei and therefore have DNA, but the red blood cells do not. The sperm cells contain most of the DNA that is in semen, but there are also some epithelial cells, which contain DNA. Much less DNA is obtained from a comparable quantity of semen that lacks sperm cells. In vaginal swabs and in buccal (inner cheek) swabs the epithelial cells have DNA. Urine has some cells in it but not too many. Sometimes enough DNA can be isolated from urine to enable successful typing, but not always.

All specimens containing nucleated cells are handled pretty much the same way in DNA analysis. The specimen is combined with an enzyme called proteinase K, which hydrolyzes the proteins nonspecifically; that is, it catalyzes the breakdown of cellular protein. Proteinase K also digests cell and nuclear membranes, releasing all the contents of the cell and its nucleus into the solution. The result of this process is called a “digest.” Next, it is common to extract DNA from this mixture using a two-phase system of phenol and chloroform. Two-phase means that these two liquids do not mix; when combined, they form two separate layers in a test tube. The two phases and the digested specimen solution are then mixed by shaking, and this causes much of the protein to move into the chloroform layer. The chloroform layer is discarded, while the phenol phase, which contains the DNA, is kept. This extraction process may be repeated two or more times.

The DNA is finally isolated from the phenol phase either by precipitation with ethyl alcohol or filtration using a miniconcentrator. DNA is insoluble in ethyl alcohol, so adding it to a DNA-containing solution causes the DNA to fall out of solution. A miniconcentrator is a small filtration device. The filter retains DNA but allows solutions of phenol, buffer, and so forth to pass through. This device can essentially wash the phenol out of the DNA. The DNA is then recovered in a buffer solution in which it is stable. The miniconcentrator procedure is now common in forensic labs.

There are other ways of processing the proteinase-K digests, such as using special columns lined with materials that adsorb DNA under certain conditions, then release it under other conditions. The mechanism of this adsorption involves oppositely charged molecules attracting one another. A positively charged molecule lining the column will bind DNA. By changing the pH of the buffer solution passing through the column, one can alter the net charges on the column molecules and the DNA so that DNA can be released from the column.