19.1: Introduction
Introduction
DNA RESTRICTION ANALYSIS
In this experiment, DNA from the bacteriophage Lambda (48,502 base pairs in length) is cut with a variety of restriction enzymes and the resulting fragments are separated using gel electrophoresis. Three samples of Lambda (phage) DNA are incubated at 37º C, each with one of the 3 restriction endonuclease enzymes: Pst1, EcoRI, and HindIII. A fourth sample will be the negative control in that is will be incubated without any endonuclease. Each of the 3 enzymes recognizes a different sequence of bases on DNA called a palindrome , and cuts within it at a specific site called a "restriction site."
The DNA samples are then loaded into wells of an agarose gel and electrophoresed, along with loading dyes (see procedure below). An electrical field applied across the gel causes the DNA fragments in the samples to move from their origin (a sample well) through the gel matrix toward the positive electrode. Small DNA fragments migrate faster than larger ones, so restriction fragments of differing sizes separate into distinct bands during electrophoresis. The loading dyes are of 2 different sizes, corresponding to very small DNA fragments and very large DNA fragments. They can be seen as the electrophoresis progresses, and they form a 'bracket' in between which the DNA fragments are moving. Otherwise, one cannot tell how far the DNA fragments have moved through the agar. The characteristic number and pattern of bands produced by each restriction enzyme are made visible by staining with a compound that binds to the DNA molecule--- methylene blue.