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Teachers Guide: Restriction Enzyme Digest Analysis
Explanation of the RFLP Technique
- Only a small segment of DNA is analyzed out of all the DNA
from the cells of an individual. This segment of DNA is used
as a probe and is analyzed from the DNA of each person in question.
(step 5).
- The DNA in each sample is digested with the same restriction
enzyme(s). Since every person has DNA with slightly different
base sequences, some of the restriction sites will be missing
or in different locations. Therefore, each person's DNA restriction
enzyme digest will produce unique DNA fragment numbers and
sizes.
- The samples of fragmented DNA are placed side by side in
an agar gel, and are then separated by size using electrophoresis.
- The double-stranded DNA fragments are chemically denatured
into single-strands ("unzipped"), and blotted onto a nylon
sheet which fixes their positions and maintains them as single-stranded
DNA.
- The nylon sheet is washed with a solution containing many
copies of a radioactive DNA probe. The probe is a very short,
single-stranded DNA molecule that will stick to ("hybridize" with)
its complementary sequence wherever it finds it among the DNA
fragments on the nylon. (This technique is called Southern
Blotting.) This further increases the selectivity of this procedure.
- Finally, an x-ray film is exposed to the nylon sheet. When
this film is developed, a dark band will appear at each location
where the probe hybridized to complementary DNA. These banding
patterns will differ from one person to another, again due
to the base pair sequence differences between peoples' DNA.
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