The Process Of Rflp Analysis
Restriction fragment length polymorphism analysis is designed to measure the length of specific segments of DNA that contain repeated patterns of base pairs. The cells containing DNA may be from a crime scene or obtained from white blood cells or a mouth swab from inside the cheek of a suspect.
1. Extract the DNA. The technician must first remove the DNA from the nucleus of the cell. Proteindigesting enzymes and detergents destroy the membranes that enclose the cell and the nucleus and free the DNA molecules. Organic solvents like phenol remove most of the non-DNA components while the DNA, which is water-soluble, remains in solution.
2. Cut it into segments. The technician now adds restriction enzymes. These are chemicals that recognize a specific sequence of base pairs, a “recognition sequence” four to six base pairs long, and cut the DNA at that point. This slices up the molecules in a controlled way. Some of the fragments will be the sought-after repeating sections.
3. Separate the fragments. Because DNA fragments have a negative electric charge, they are attracted toward a positive electrode when the technician deposits them on a porous gel. The shorter fragments move out in front of the longer ones before the current is turned off. The pieces of DNA are now aligned into bands according to length. As a standard, fragments of known length are included, and they form comparison bands along the edge of the gel.
4. Transfer to a membrane. The DNA is drawn out of the gel onto a nylon membrane and dried. This step stabilizes the alignment of the fragments. At this point the technician also “unzips” the DNA by means of a strong alkali, producing separate strands.
5. Wash the fragments with probes. These sections of synthetic DNA are complements of the particular repeating section that the investigator wants to measure. They will bind with that section and not with other DNA fragments. Each probe has a marker, a radioactive isotope, built into it.
6. Visualize the array. So far the DNA fragments are invisible. The technician now washes the membrane to remove the excess probes, dries it, and lays it on a sheet of X-ray film. The radioactive atoms attached to the probes darken the film wherever the probes are concentrated.
7. Analyze the results. By comparing the location of this darkening with the test samples of known length, the investigator determines the length of the fragments in the target section.