RESTRICTION OF LAMBDA DNA

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ANALYZING PRE-CUT DNA

One of the basic tools of modern biotechnology is gene splicing or recombinant DNA technology. The basic concept behind gene splicing is to remove a fragment of DNA from one organism and combine it with the DNA of another. The result would be the host organism implementing the instructions provided by the newly inserted DNA fragment. For example, certain plants can be given the genes for resistance to pests or disease.

The first step in recombinant DNA technology is to locate the specific gene of interest on a chromosome. A restriction enzyme is then used to cut (restrict) the targeted gene from the rest of the chromosome. This same enzyme will then be used to cut the DNA into which the fragment will be inserted.

The ability to cut (restriction) DNA predictably and precisely enables biotechnologists to readily manipulate and recombine DNA molecules. Restriction of DNA is used in recombinant DNA technology, DNA fingerprinting, DNA sequencing, and many other applications in the field of biotechnology. The restriction enzymes which are used to cut DNA are found to occur naturally in bacteria. They are in fact a means by which bacteria protect themselves from any foreign DNA which might invade the bacterial cell. Any foreign DNA encountering a restriction enzyme would be cut into fragments and rendered ineffective.

Each restriction enzyme is specific, and cuts at only a very specific sequence of the DNA molecule. For example, EcoRI will cut only at: GAATTC If this specific

CTTAAG

sequence occurs in more than one location on a DNA molecule, EcoRI will cut all of these sites. Therefore if a given piece of DNA (linear) is cut with a restriction enzyme, whose specific code is found at 5 different locations on the DNA molecule, the result will be 6 fragments of varying lengths. The length of each fragment would depend upon the location of restriction sites on the DNA molecule.

DNA which has been cut with restriction enzymes can be visualized using a process known as agarose gel electrophoresis. The term electrophoresis means to carry with electricity. Agarose gel electrophoresis separates DNA fragments by molecular weight. DNA fragments are loaded into an agar block, which is placed into a chamber filled with a liquid buffer soltion. A direct current is passed between wire electrodes at each end of the chamber. DNA fragments are negatively charged, and when placed in an electric field will be drawn toward the positive pole and repelled by the negative pole. The matrix of the agarose gel acts like a sieve through which smaller DNA fragments can move more easily than larger ones. Therefore, the distance and rate at which DNA fragments migrate through the gel is inversely proportional to its molecular weight. Over a period of time smaller fragments will have traveled further than larger ones. Fragments of the same size stay together and pool into discrete "bands".

Genetic Engineering

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Electrophoresis of lambda DNA samples cut using 3 different restriction enzymes is shown in figure 1. Notice that in each case the same lambda DNA was used, however a different restriction enzyme (EcoRI, BamHI, and Hind III) was used each time to produce a unique pattern of fragments and banding in each lane. The relative size of fragments contained in each band can be determined by how far each band has traveled from the origin (well). For example, the HindIII enzyme has produced the smallest fragments (two bottom bands).

Figure 1.

Lane No.

Enzyme

Genetic Engineering

Science

TL 2-2