Teachers Guide: Restriction Enzyme Digest Analysis

One very new and interesting application of recombinant DNA technology is in the area of forensic science, in which scientists use restriction fragment length polymorphism (RFLP) analysis to help solve rape, murder, and paternity cases. New York, California, and Florida are among the states that currently allow DNA evidence to be used in courts of law. Soon the United States Supreme Court may be asked to rule on the validity of DNA analysis as admissible evidence.

In 1984 British researcher Alec Jeffreys discovered a new technique for analyzing DNA differences between people. He coined the term "DNA fingerprinting". His new method, if properly applied, would produce DNA analyses that are so accurate, the chance of two people having the same such "fingerprint" is 1 in 3 billion to 30 billion.

Virtually every cell in our body contains DNA. Every individual has unique DNA and this results in each of us having a unique look - different color eyes or hair, perhaps a nose that's longer or shorter.

Interestingly, only about 5% of our DNA actually codes for functional genes. Between the pieces of coding DNA are pieces of DNA with no known function. There are slight differences in base pair sequences in both the coding and non-coding DNA between one individual and the next. Jeffreys' method made use of these differences so that DNA from an individual would produce a distinct pattern when chemically treated in a certain manner. This pattern can be used to identify a person much like their fingerprint.

Purpose

This lab exercise is designed to simulate RFLP analysis. The key word here is "simulate". The procedure has been altered for reasons of simplicity, safety and cost effectiveness. We will not be using radioactive probes. Instead we will visualize all of the fragments by staining. In addition, if the lab is followed as written, the students will assume that they are analyzing 3 different DNA samples using the same restriction enzyme. What they will actually be doing is analyzing the digestion fragments of the same DNA sample (lambda phage) using 3 different restriction enzymes (EcoRI, BamHI, and HindIII). The important point is the comparison between the DNA banding pattern of the "suspect" and the "murderer".

It is anticipated that, prior to this activity, the students will have a basic understanding of DNA structure, restriction enzyme function, and gel electrophoresis as well as a cursory introduction to restriction fragment length polymorphisms (RFLPs).

 

RFLP Analysis Learner Outcomes

This lab simulation can be applied many different ways depending upon the content and focus of your curriculum. Some possible goals and objectives are listed below.

Each student should:

1. gain a better understanding of one of the applications of DNA analysis.
2. become more familiar with the function of restriction enzymes.
3. better understand the theory behind the gel electrophoresis process.
4. become proficient at running gel electrophoresis equipment.
5. know what RFLP's are and how they relate to DNA analysis.

Explanation of the RFLP Technique

1. 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).
2. 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.
3. The samples of fragmented DNA are placed side by side in an agar gel, and are then separated by size using electrophoresis.
4. 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.
5. 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.
6. 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.