1. Restriction enzymes
A. In nature, bacteria use restriction enzymes to cut foreign DNA, such as from viruses or other bacteria.
B. Restriction enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at a specific point in these sequences.
C. Each restriction enzyme cleaves a specific sequence of bases called a restriction site.
1. These are a series of four to eight bases on both strands running in opposite directions.
2. If the restriction site on one strand is 3�-CTTAAG-5�, the complementary strand is 5�-GAATTC-3�.
D. Because the target sequence usually occurs (by chance) many times on a long DNA molecule, an enzyme will make many cuts.
2. Restriction fragment analysis is used in DNA fingerprinting
A. Restriction fragment analysis indirectly detects certain differences in DNA nucleotide sequences.
B. After treating long DNA molecules with a restriction enzyme, the fragments can be separated by size via gel electrophoresis.
1. This produces a series of bands that are characteristic of the starting molecule and that restriction enzyme.
C. Gel electrophoresis
1. Used any time someone has a tube of cut pieces of DNA of various sizes that must be separated.
2. Mix gel powder with buffer and ethidium bromide and pour into mold, allow to solidify.
3. Place gel in gel running box.
4. Mix DNA samples with loading dye (loading buffer) and load into gel wells.
5. Place (+) electrode at bottom of gel and ( -) electrode at top.
6. Set voltage at 50-200 volts.
7. Run gel.
a. DNA fragments migrate through gel at rates based on size of spaces between gel molecules.
b. Large DNA fragments move more slowly than shorter ones.
8. Place gel on U.V. light box.
9. Cut band of DNA of correct size out of gel and chemically extract DNA from surrounding gel fragment (if needed).
D. We can use restriction fragment analysis to compare two different DNA molecules representing, for example, 2 different suspects in a crime.
1. Because the DNA molecules must differ slightly in DNA sequence, they may differ in one or more restriction sites.
2. If they do differ in restriction sites, each will produce different-sized fragments when digested by the same restriction enzyme.
3. In gel electrophoresis, the restriction fragments from the two people will produce different band patterns.
3. DNA fingerprinting
A. In violent crimes, blood, semen, or traces of other tissues may be left at the scene or on the clothes or other possessions of the victim or assailant.
B. DNA testing can identify the guilty individual with a high degree of certainty, because the DNA sequence of every person is unique (except for identical twins).
C. Restriction fragment analysis can detect similarities and differences in DNA samples and requires only a tiny amount of blood or other tissue.
D. In practice, forensic DNA tests focus on only about five tiny regions of the genome.
1. The probability that two people will have identical DNA fingerprints in these highly variable regions is typically between one in 100,000 and one in a billion.