Download Restriction Enzymes - Seattle Central College

Restriction Enzymes
• Restriction enzymes are bacterial enzymes that cleave the sugar-phosphate backbone
of the DNA at specific nucleotide.
• They are member of the class of nucleases. Endonucleases cleave nucleic acid at
internal positions, while exonucleases progressively digest from the ends of the
nucleic acid molecules.
• The three dimensional structure of the restriction enzyme allows it to fit perfectly in
the grove formed by the two strands of DNA molecule. When attached to the DNA,
the enzyme slides along the double helix until it recognizes a specific sequence of
base pairs which signals the enzyme to stop sliding. The enzyme, then digest the
DNA at that site.
• If the a specific site occurs in more than one location on a DNA molecule, a
restriction enzyme will make a cut at each of those sites, resulting in multiple
fragments of DNA
• Each restriction enzyme recognizes a specific nucleotide base sequence, termed
palindrome, in the DNA called restriction site.
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The cutting produces two DNA fragments. The staggered termini are called sticky
ends because they can reassociate with another by hydrogen bonding.
Restriction enzymes can be used in combination to digest large DNA molecules into
smaller fragments. Because the enzyme always cuts at the same site, DNA from a
particular molecule will generate a reproducible set of fragments.
Advantages of restriction enzymes:
a. Each has only restriction activity.
b. Each cuts in a predictable and consistent manner.
c. They require only magnesium ion as a cofactor; no ATP is
needed.
DNA
• The DNA digested by the restriction enzymes used in the experiment is Lambda
DNA, which comes from bacterial virus, or bacteriophage.
• Bacteriophage attacks bacteria by inserting its nucleic acid into the host bacteria
which replicates rapidly inside host cells until cells burst. The released phage
carries out the infection in other bacteria.
3. Agarose Gel Electrophoresis
• The fragments of DNA can be visualized through gel electrophoresis.
• Because the of the negative charge of the phophoryl group, the DNA will move away
from the negative electrode and migrate to the positive electrode.
• The smaller fragments will move more rapidly than the larger ones.
• The sizes of each fragment can be determined by comparison with migration patterns
of DNA fragments of known size. This is your DNA standard.
Experiment
Wipe your table with 70% ethanol or a bacteriacide before and after the
experiment. Wash you hands before leaving the lab.
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There are only 8 electrophoresis apparatus, so there might some groups with three
members or 4 members.
There are 8 wells available to use, there is room to practice loading
Each pair or partner will turn in separate lab reports.
Agar Preparation
• Make sure the agar is completely dissolved.
Sample Preparation
• Keep the digests in ice until you are ready to load.
• Cover agar and the bottom of the box completely with the 1x TAE buffer. #6.
Practice loading in lane 8 using the practice solution prepare in #2.
• Run the gel until the tracking dye reaches the middle of the top red strip.
• While the gel electrophoresis is running run the size exclusion experiment.
Staining:
This is the touchiest part of the experiment. Follow the instructions closely.
Get organized with the warm water. The staining time is crucial. It can not be more than
3 minutes.
Data Analysis
• The DNA standard is the marker DNA. If all goes well with the experiment you
should have 7 bands. Measure the distance of the bands starting from the wells.
Note the pattern of the bands and compare it pattern on the lecture notes.
• Once the bands are measured, a standard curve of distance vs. bp is graphed. You
might have to rescale the x axis to accommodate your distance travelled
• From the standard curve the number of base pairs cleaved by the HindIII, PsfI and
EcoRI and the lambda DNA are determined.