What is restriction fragment analysis? Restriction fragment analysis is a process used to compare the DNA of two or more different organisms.

There are 4 processes involved in restriction fragment analysis: Electrophoresis Blotting Hybridisation Autoradiography

Gel Electrophoresis Two or more samples of DNA are separated by gel electrophoresis. Each will form a characteristic pattern of bands.

Blotting A sheet of nitrocellulose paper (filter paper) is pressed onto the gel. Capillary action will pull the separated fragments onto the paper, in such a way that they are positioned in bands exactly as on the gel.

Hybridisation The paper blot is exposed to a solution that contains radioactive labeled probe. The probe is single stranded DNA complementary to the DNA sequence of interest, and it will pair complementarily with the separated fragments.

Autoradiography A sheet of photographic film is laid on the paper. The radioactive probe will cause images to show up at places where there are DNA fragments. These images can be compared to tell the difference between the samples of DNA being analyzed.

Restriction Fragment Analysis Procedure Step 1 : Restriction fragment preparation – DNA is extracted from white blood cells taken from 3 individuals. Individual I is known to carry a disease allele. A restriction enzyme is added to the 3 samples of DNA to produce the restriction fragments. Page 241, Figure C of your textbooks! I II III 1

Step 2 : Gel Electrophoresis – The mixture of restriction fragments from each sample of DNA is separated by electrophoresis. Each sample forms a characteristic pattern of bands. I II III 2

Step 3 : Blotting – the DNA bands are treated to separate the strands of the molecules, and the single strands are transferred by simple blotting onto special filter paper. 3

Step 4 : Radioactive Probe – the paper blot is immersed in a solution of a radioactive probe, a single-stranded DNA molecule that us complementary to the DNA sequence of the genetic marker of interest. The probe attaches by base-pairing to restriction fragments arising from the marker DNA. 4

Step 5 : Detection of radioactivity (autoradiography) – the unattached probe is rinsed off, and a sheet of photographic film is laid over the paper. The radioactivity in the probe exposes the film to to form an image corresponding to specific bands-the bands containing DNA that base pairs with the probe. 5

Benefits of Restriction Fragment Analysis With the use of restriction fragments, a molecular geneticist can show how unique one’s DNA is to everyone else, and show how no two people are unlike (unless you have an identical twin). It can be used to show traits that are passed on through your family. Restriction fragment analysis is used in research of human genetics.

Restriction fragment analysis has a practical application in that it can be used to detect which allele patterns are normal or harmful in your family, and then can be used to test any family members who may be carriers of that disease.

Ethical Problems? There are no ethical problems dealing with restriction fragment analysis that I have come across.

Bibliography Biology Textbook, Fourth Edition - Chapter 12.11, pages