Tuesday, May 17, 2011

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.

Separation, Labeling, and Detection of DNA Segments

Suppose a scientist were looking for a certain sequence pattern in a mixture of DNA fragments of different sizes. The mixture could be separated by electrophoresis on an agarose gel and then fixed on a membrane by Southern blotting. Then, in order to locate the desired DNA sequence pattern on the blot, a single-stranded segment of DNA called a “probe” could be added directly to the blot.

A probe is a single strand of DNA with a sequence complementary to the target strand of DNA. There are methods to easily label the probe—and thus visualize the target sequence that the probe binds with—using radioactive phosphorus-32 (32P) labels. Once the probe is labeled, it can be placed in a solution with the membrane-containing mixture of all the separated DNA fragments. Under controlled conditions the probe will base pair only with the exactly complementary sequence; this process is called “hybridization.” The reason that the probe can base pair (hybridize) with its complementary sequence directly on the membrane is that the Southern blotting process has already denatured the DNA on the membrane, that is, rendered it single stranded.

The excess probe and solution can then be washed away, leaving the membrane with a radioactive probe adhering to one DNA fragment. The scientist can now visualize the target DNA sequence by placing this membrane against a sheet of X-ray film. The radioactivity exposes the film just as X-rays would. When the film is developed, it shows an image of the DNA fragment that had the radioactive label on it. This process, illustrated in the diagram, is called “autoradiography.” The first forensic DNA typing was done in this way. But radioactive materials such as 32P pose a health hazard, so it is expensive and cumbersome to handle and store them in the safe, legal way. Later, a method using light-emitting (chemiluminescent) probes was developed so scientists could avoid having to work with the radioactive phosphorus.

As already discussed, scientists can separate DNA fragments of different sizes using electrophoresis on agarose gels, but the electrophoresis can also be carried out in tiny capillaries that are filled with a solution of buffer and polymers (somewhat comparable to agarose). This process is capillary electrophoresis (CE) and has some significant advantages over traditional gel electrophoresis. One is that heat does not build up as much in the capillary. Heat buildup is a problem in electrophoresis, both because it can denature protein or DNA molecules and because it can change the properties of the medium (the agarose or buffer solution) in a way that affects the migration properties of the molecules. If heat buildup is not controlled, it can affect the reproducibility of electrophoresis.

 DNA probes and Southern blotting. Probe:
Short sequences of single-stranded DNA, 20-50 nucleotides (building blocks) long, can be chemically synthesized and them made radioactive or fluorescent. This DNA can base pair with a complementary DNA or RNA strand, labeling it as either radioactive or luminescent. Southern blotting:
1. The DNA is fragmented by the addition of a restriction enzyme (biological catalyst that cuts DNA at specific sites).
2. The fragments are next separated, according to their length, into invisible bands on electrophoresis gel.
3. The separated DNA fragments can then be transferred to a nitrocellulose or nylon
membrane. The DNA molecules are made to stick permanently to the membrane.
4. When a radioactive DNA probe solution is added to the membrane and then gently washed off, some of the probes will remain attached to any DNA fragments that have a complementary sequence.
5. The membrane is placed against a photographic film. 6. The film darkens wherever the radioactive DNA probe has bound to the membrane. The probe has identified which DNA fragment contains the complementary DNA sequence for the probe.