Download DNA Fingerprinting (RFLP Analysis) Introduction DNA fingerprinting

DNA Fingerprinting (RFLP Analysis)
Introduction
DNA fingerprinting is a technique that is used to identify patterns that occur in DNA. No two
organisms have identical DNA so this procedure can be used to identify if a sample of DNA
came from a particular individual.
We will use this procedure in lab to identify whether a sample of DNA found at a crime scene
belongs to one of three suspects.
The technique has a variety of other uses such as
being used to identify whether individuals carry genes for certain genetic diseases.
Restriction Enzymes
The technique of DNA fingerprinting requires that the DNA be cut up into small fragments.
Restriction enzymes are used to perform this digestion.
Restriction enzymes were discovered in bacteria, which use them as a defense mechanism to cut
up the DNA of viruses or other bacteria.
Hundreds of different restriction enzymes have been isolated. Each one cuts DNA at a specific
base sequence. For example, EcoRI always cuts DNA at GAATTC as indicated below.
The sequence GAATTC appears three times in the DNA strand below. As a result, the strand is
cut into four pieces.
Other restriction enzymes cut at different sites, some examples are listed below.
Enzyme
Bam HI
Hae III
Pst I
Hinf I
Cutting Site
GGATCC
GGCC
CTGCAG
GANTC
In RFLP analysis, the DNA of an organism is cut up into fragments using restriction enzymes. A
large number of short fragments of DNA will be produced.
Restriction enzymes always cut at the same base sequence. Because no two individuals have
identical DNA, no two individuals will have the same length fragments. For example, the
enzyme EcoRI always cuts DNA at the sequence GAATTC. Different people are going to have
different numbers of this particular sequence and will therefore have different fragment lengths.
In addition, some of them will be at different locations on the chromosome.
Gel Electrophoresis
Electrophoresis is a technique used to separate the DNA fragments according to their size. They
are placed on a sheet of gelatin and an electric current is applied to the sheet. DNA is charged
and will move in an electric field toward the positive pole.
In the diagram below, holes (wells) in the gelatin can be seen. DNA samples placed in these
wells will migrate through the gelatin toward the + side after an electric current is applied.
The smallest fragments will move the fastest because they are able to move through the pores in
the gelatin faster. Bands will be produced on the gelatin where the fragments accumulate. The
shortest fragments will accumulate near one end of the gelatin and the longer, slower-moving
ones will remain near the other end.
In the diagram below, four samples of DNA were placed on the gelatin. After an electric current
was applied for a period of time, the fragments separated. Notice that sample D on the right does
not match the other three samples.
The DNA bands must be stained to make them visible. Ethidium bromide-stained DNA will
fluoresce when illuminated with UV light.
PCR techniques are used to produce sufficient quantities of DNA for this technique.