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Restriction Fragment Length Polymorphisms (RFLP's)


Autopsies | Blood Analysis | Absorption-elution technique | Kastle-Meyer Colour Test | Luminol Test | Human or Not? Precipitin Test | Blood stain patterns | DNA Fingerprinting | Restriction Fragment Length Polymorphisms (RFLP's) | Short Tandem Repeats (STR) | Entamology | Common Insects | Links


D.N.A. Fingerprinting

DNA profile analysis was first made possible by the use of Restriction Fragment Length Polymorphisms (RFLP). Though it
sounds complicated, RFLP is based on simple concepts. It is made possible by special 'molecular scissors' called restriction
enzymes. Restriction enzymes are molecules found in bacteria, which cut DNA at specific sites. There are many different types
of restriction enzymes and each type recognizes a different site on the DNA chain. For example, the enzyme TaqI cuts the
DNA double strand everywhere it sees the sequence TCGA. In bacteria these enzymes are present to destroy foreign DNA, such
as might be present from an attacking virus.
In DNA profiling by RFLP analysis, scientists use specific restriction enzymes to cut STRs out of the DNA molecules. Scientists
use restriction enzymes that cut roughly on either side of the STRs they are trying to find. In practice, usually two or three
different restriction enzymes are used, so that enough sequences are isolated The final product of this stage is a test-tube of
DNA fragments, which include both the desired STR sequences and the rest of the genome. The only thing left to do is isolate
the STRsequences (restriction fragments) and determine how many times each sequence repeats (length polymorphisms).
To accomplish this there is yet another technique, electrophoresis. 
In electrophoresis, we inject a few microliters of a DNA sample into tiny wells at one end of a rectangular gel.
On the same end of the gel we place a negative electrode; on the opposite end, we place a positive electrode. When we turn on
the electrodes, the negatively charged DNA fragments are pulled across the gel by the positive electrode.As you might expect,
the shorter DNA fragments, because of their smaller size, travel faster than longer DNA fragments. After a certain amount of
time, the DNA fragments are distributed by size (length) along the gel, with the smallest fragments near the positive electrode
and the larger fragments near the negative electrode.


Once the gel has been "run," the only task remaining is to illuminate the DNA fragments. With PCR, the entire gel is simply

soaked in a staining solution. The DNA fragments appear as little bands on the gel and we can determine their approximate

length by their position on the gel. To illuminate the DNA profile from an RFLP analysis, we first need to expose it to

radioactively labeledprobes, which specifically target the STR sequences that we are trying to find. Then we can expose

it to an X-ray film, which willvisualize only the fragments that were detected by the radioactively labeled probes. As long

as the same techniques are used inpreparing all the samples in a test, a direct comparison of the DNA profiles is an

accurate way to match identities or confirmrelationships. A person's DNA profile will be approximately a 50/50

combination of his or her mother and father. An exact DNAprofile match either indicates that both samples were

taken from the same person, or from that person's identical twin.