NAME: ________________________________
Digesting DNA Using Restriction Enzymes
PURPOSE
To learn how to set up DNA digestions with restriction enzymes in order to
create DNA fingerprints for analysis. To gain an understanding of restriction
endonucleases critical for genetic engineering and biotechnology.
INTRODUCTION
As you know the DNA molecule can be compared with a zipper that can opened
up to allow replication and transcription. Scientists have found that there are
many bacterial enzymes called restriction endonucleases or simply restriction
enzymes that cut both strands of the DNA molecule at one specific site in the
nucleotide sequence. This is similar to cutting a zipper into pieces by cutting
only between certain teeth of the zipper. The restriction endonucleases cut the
DNA backbone at specific sequences called recognition sites that are usually 4
or 6 base pairs long. Each restriction enzyme recognizes a different recognition
sequence. As a result, a single piece of DNA is cut into several DNA fragments
of different sizes. Where the DNA is cut depends on its base sequence, so that
different pieces of DNA are cut into different pieces. The endonuclease
"restricts" the size of the fragments. . These DNA fragments arising from
different pieces of DNA can then be analyzed by comparing the patterns of
bands observed after agarose gel electrophoresis, a process that separates DNA
fragments based on their size. Restriction enzyme digestion is also central to
genetic engineering and gene cloning procedures.
MATERIALS
Latex Gloves
Safety Glasses
Restriction Enzyme
Universal Buffer
Sterile Water
Acetylated Bovine Serum (BSA)
Permanent Marker
Microcentrifuge Tube Rack
Microcentrifuge Tubes of Suspect DNA
Microcentrifuge Tube Crime Scene DNA
0.5-10 L Micropipetter
20-200 L Micropipetter
Micropipette Tips
Container for Tip Disposal
PROCEDURE
1.
Before beginning be sure to glove your hands and put on safety glasses.
USEFUL TIP: all the reagents for this experiment have different
colors; this will aid you during the pipetting process. Remember to
check your micropipette tip and to make sure you are pipetting the
right volume.
2.
In a microcentrifuge tube rack locate the following stock microcentrifuge
tubes:





DNA Samples (suspect DNA and crime scene DNA)
Sterile Water
Restriction Enzyme
Universal Buffer
BSA 1:10 dilution
3.
Using the 20-200 L micropipetter place 26 L sterile water into each of
the tubes containing sample DNA.
4.
Using the 0.5-10 L micropipetter place 4 L universal buffer into each of
the sample tubes.
5.
Using the 0.5-10 L micropipetter place 4 L BSA into each sample tube..
6.
Using the 0.5-10 L micropipetter place 5 L of restriction enzyme into
each sample tube.
7.
Gently! Tap each of the sample tubes using the tip of your finger for 20
seconds; this will mix the reaction cocktail. Try not to splash and splatter
the solution in the tube. Do not shake or invert the microfuge tubes!
8.
Incubate the sample tubes overnight at room temperature.
9.
The restriction digests are ready for analysis by agarose gel
electrophoresis. They can be stored frozen until ready for electrophoresis.
.
QUESTIONS AND CONCLUSIONS
1.
Name some measures that you took to prevent contamination of your DNA
samples during this lab.
2.
What is DNA fingerprinting?
3.
State a situation other than crime scene analysis where DNA
fingerprinting can be useful.
REFERENCES
1. Glencoe Biology, The dynamics of life
2. Sambroock, Molecular Biology
3. Modern Biology