Biology 141 Laboratory 5
Spring 2014
Introduction to the use of Restriction Enzyme Analysis
Special restriction enzymes have been discovered in many different bacteria and other single-celled organisms. These restriction enzymes are able to read along a length of DNA and find a particular sequence of bases that they recognize. This recognition sequence is generally from 4 to 6 base pairs in length. Once the sequence is located, the enzyme will attach to the DNA molecule and cut each strand of the double helix. The restriction enzyme will continue to do this along the full length of the DNA molecule cutting it into fragments.
The use of restriction enzymes has been important for modern forensic analysis of DNA evidence.Although 99.8% of DNA coding for genes is the same in humans, the noncoding DNA sequences are more variable between persons. This means enough of the DNA is different to distinguish one individual from another, unless they are identical twins. DNA fingerprinting (or profiling) uses repetitive DNA sequences that are highly variable. These sequences are so variable that unrelated individuals are extremely unlikely to have the same patterns across multiple “variable tandem repeat” regions. However, they are more likely to be similar between closely related humans. To increase the probability of correctly identifying an individual person by using DNA fingerprinting, it is essential to scoremultiple regions for accurate analysis.
In this experiment, we will perform 3 restriction digestions of Lambda DNA (a common virus that infects bacteria). We are using the Lambda DNA as a substitute for actual crime scene DNA. We will be using the restriction enzymes called EcoR1, HindIII, and PST1 (bacterial sources Escherichia coli, Haemophilusinfluenzae, and Providenciastuartii, respectively). After the digestion, in the following weeks lab, we will separate the unique DNA fragments produced by each restriction enzyme using gel electrophoresis, a process that involves application of an electric field to cause the DNA fragments to migrate through an agarose gel, separating them by size. We will then use the results to complete a forensic analysis.
Restriction Digests:
1. Put on gloves. Keep tubes on ice through step 4.
2. Label 3 microfuge tubes EcoRI, HindIII, PST1 and add a symbol for your group so that you can identify your tubes later.
3. Add reagents to each tube in the following order: (check final volume in each to make sure they all look the same!)
Tube: / EcoRI / HindIII / PST1Water / 28.0 µl / 28.0 µl / 28.0 µl
10X buffer / 4.0 µl / 4.0 µl / 4.0 µl
Lambda / 4.0 µl / 4.0 µl / 4.0 µl
Enzyme / EcoRI4.0 µl / HindIII4.0 µl / PST14.0 µl
Total / 40.0 µl / 40.0 µl / 40.0 µl
4. Tap each tube gently with your finger to thoroughly mix.
5. Put your tubes in a water bath rack and place in 37o C bath for one hour.
6. Remove tubes, place in a microfuge rack.
7. Your instructor will put the tubes in 4 o C till next week.