DNA Fingerprinting

DNA Fingerprinting A technique used to distinguish between individuals of the same species using only samples of their DNA. Two humans will have the vast majority of their DNA sequence in common. Genetic fingerprinting exploits highly variable repeating sequences called mini-satellites. Two unrelated humans will be likely to have different numbers of mini-satellites at a given point.

Identical Twins and DNA http://www.youtube.com/watch?v=3qqrvrSkrGU

Genetic fingerprinting is used in forensic science, to match suspects to samples of blood, hair, saliva or semen. It has also led to several exonerations of formerly convicted suspects.

It is also used in such applications as identifying human remains, paternity testing and to match organ donors.

David Milgaard Canadian who was wrongfully convicted for the murder and rape of nursing assistant Gail Miller. He was released in 1999 and compensated (10 million) after spending 23 years in prison. DNA evidence based on DNA fingerprinting lead to his exoneration.

Why can we use DNA to identify people? 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.

Identifying Patterns 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.

Obtaining DNA Samples Testing is subject to the legal code of the jurisdiction in which it is performed. Usually the testing is voluntary, but it can be made compulsory by such instruments as a search warrant or court order. Several jurisdictions have also begun to assemble databases containing DNA information of convicts.

DNA in the United Kingdom The United Kingdom currently has the most extensive DNA database in the world, with well over 2 million records as of 2005. The size of this database, and its rate of growth, is giving concern to civil liberties groups in the UK, where police have wide-ranging powers to take samples and retain them even in the event of acquittal.

Steps in Extracting DNA 1) DNA fingerprinting begins by extracting DNA from the cells in a sample of blood, saliva, semen, or other appropriate fluid or tissue.

2) RFLP (restriction fragment length polymorphism) analysis is performed by using a restriction enzyme to cut the DNA into fragments. A restriction enzymeis an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the phosphate backbones of the double helix without damaging the bases.

Rather than cutting DNA indiscriminately, a restriction enzyme cuts only double-helical segments that contain a particular nucleotide sequence, and it makes its incisions only within that sequence--known as a "recognition sequence"--always in the same way. Most enzymes make slightly staggered incisions, resulting in "sticky ends", out of which one strand protrudes

Two examples of restriction enzymes are EcoRI and HindIII. These enzymes recognize specific sequences of DNA and cut the DNA at that point.

EcoRI and HindIII Enzyme recognition sequence cut EcoRI 5'GAATTC 3'CTTAAG 5'---G AATTC---3' 3'---CTTAA G---5' HindIII 5'AAGCTT 3'TTCGAA 5'---A AGCTT---3' 3'---TTCGA A---5'

The cut pieces of DNA are separated into bands during agarose gel electrophoresis. Larger bands move slower, while smaller bands move faster. This creates what is called a ‘banding pattern’.

Next, the bands of DNA are transferred via a technique called Southern blotting.

Preparing A Southern Blot 1. Isolate 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 3. Sorting the DNA pieces by size. The process by which the size separation 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.

6) In order to analyze a Southern Blot, a radioactive genetic probe is used. 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 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.

Southern Blot Video http://www.youtube.com/watch?v=VmCTsmhx2_w