Recombinant DNA Labs: Basic Tools For The Molecular Biologist

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).