Making a Radioactive Probe

1. Obtain some DNA polymerase [pink]. Put the DNA to be made radioactive (radiolabeled) into a tube.

2. Introduce nicks, or horizontal breaks along a strand, into the DNA you want to radiolabel. At the same time, add individual nucleotides to the nicked DNA, one of which, *C [light blue], is radioactive.

3. Add the DNA polymerase [pink] to the tube with the nicked DNA and the individual nucleotides. The DNA polymerase will become immediately attracted to the nicks in the DNA and attempt to repair the DNA, starting from the 5' end and moving toward the 3' end.

4. The DNA polymerase [pink] begins repairing the nicked DNA. It destroys all the existing bonds in front of it and places the new nucleotides, gathered from the individual nucleotides mixed in the tube, behind it. Whenever a G base is read in the lower strand, a radioactive *C [light blue] base is placed in the new strand. In this fashion, the nicked strand, as it is repaired by the DNA polymerase, is made radioactive by the inclusion of radioactive *C bases.

5. The nicked DNA is then heated, splitting the two strands of DNA apart. This creates single-stranded radioactive and non-radioactive pieces. The radioactive DNA, now called a probe [light blue], is ready for use.

Southern Blot

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The Southern Blot is one way to analyze the genetic patterns which appear in a person's DNA. Performing a Southern Blot involves:1. Isolating the DNA in question from the rest of the cellular material in the nucleus. This can be done either chemically, by using a detergent to wash the extra material from the DNA,or mechanically, by applying a large amount of pressure in order to "squeeze out" the DNA.
2. Cutting the DNA into several pieces of different sizes. This is done using one or more restriction enzymes.
3. Sorting the DNA pieces by size. The process by which the size separation, "size fractionation," is done is called gel electrophoresis. The DNA is poured into a gel, such as agarose, and an electrical charge is applied to the gel, with the positive charge at the bottom and the negative charge at the top. Because DNA has a slightly negative charge, the pieces of DNA will be attracted towards the bottom of the gel; the smaller pieces, however, will be able to move more quickly and thus further towards the bottom than the larger pieces. The different-sized pieces of DNA will therefore be separated by size, with the smaller pieces towards the bottom and the larger pieces towards the top.
4. Denaturing the DNA, so that all of the DNA is rendered single-stranded. This can be done either by heating or chemically treating the DNA in the gel.
5. Blotting the DNA. The gel with the size-fractionated DNA is applied to a sheet of nitrocellulose paper, and then baked to permanently attach the DNA to the sheet. The Southern Blot is now ready to be analyzed.
In order to analyze a Southern Blot, a radioactive genetic probe is used in a hybridization reaction with the DNA in question (see next topics for more information). If an X-ray is taken of the Southern Blot after a radioactive probe has been allowed to bond with the denatured DNA on the paper, only the areas where the radioactive probe binds [red] will show up on the film. This allows researchers to identify, in a particular person's DNA, the occurrence and frequency of the particular genetic pattern contained in the probe.

What is DNA Fingerprinting?

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The chemical structure of everyone's DNA is the same. The only difference between people (or any animal) is the order of the base pairs. There are so many millions of base pairs in each person's DNA that every person has a different sequence.
Using these sequences, every person could be identified solely by the sequence of their base pairs. However, because there are so many millions of base pairs, the task would be very time-consuming. Instead, scientists are able to use a shorter method, because of repeating patterns in DNA.
These patterns do not, however, give an individual "fingerprint," but they are able to determine whether two DNA samples are from the same person, related people, or non-related people. Scientists use a small number of sequences of DNA that are known to vary among individuals a great deal, and analyze those to get a certain probability of a match.

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