In this chapter: >> Recombinant DNA - Biotechnology - Monohybride crossings - Cloning

For al long time people have tried to change the characteristics of bacteria (see the chapter about bacteria) by cutting and pasting in the DNA.
The discovery of the so called restriction-enzymes was an important progress.
Certain viruses, called bacteriophages, can infect certain bacteria and can't infect others. This restriction is caused because the bacterium makes enzymes which can break down the DNA of the bacteriophage. The parts that cause this restriction are called restriction-enzymes. Restriction enzymes break down specific parts of the DNA. The bacterium produces methyl groupes in his own DNA as protection to prevent his own DNA from being broken down.
A restriction enzyme only breaks down the parts that have a specific sequence of adenine, thymin, cytosine and guanine. For instance it only cuts out the pieces with base-sequence G-A-A-T-T-C. This way you can cut very precisely. The pasting is done by a virus, which inplants his DNA in the DNA of the bacterium.

So these enzymes process the DNA of a bacteriophage and they also process the DNA of the bacterium itself in some unprotected places. This can cause an open part in the circular DNA and the new virus DNA can settle itself in the open part.
This way people can manipulate the bacterium anyway they want by using the right bacteriophage. However, humans are made up of far more cells, which makes this manipulating more difficult.

Virusinfection

MOLECULAR CELL BIOLOGY 2/E by Darnell, Lodish and Baltimore © 1990 by Scientific American Inc. Used with permission of W.H. Freeman and Company.

The cutting and pasting of the DNA was made more easy in 1976 when the university of Edinburgh developed a very sensitive method (method of Southern) to show the genes and parts of them. This technique makes it possible to split up DNA and connect the pieces to other (new) DNA pieces.

This new molecule is called a hybrid molecule. That's why the method is often reffered to as hybridization.

Location of specific genes in the method of Southern goes as follows:

The plasmides are first cut in pieces by restriction enzymes
The pieces are placed in a gel. The gel is exposed to an electric voltage. As a result the pieces of DNA are thrown apart depending on their size. Small pieces will go down and large pieces will go up.
The gel is manipulated with lye. As a result the strands of DNA will unfold themselves.
The gel is put on a cellulose nitrate filter with a dry piece of filtering paper on top. The dry paper will absorb the water from the gel. The pieces of DNA will move with the water, but will be stopped by the cellulose nitrate filter.
The filter with the pieces of DNA is put in a bath with a radioactive gene. This gene will only connect itself to the DNA at complementary places. These connections can be made visible by putting the gel on top of a photographic film. This way it will be easy to detect the DNA that has absorbed the radioactive genes. By the way the complementary structure does not need to be exact. Detection of genes is already possible if the resemblance is 60% or more.

So it became possible with the method of Southern to locate the position of specific genes. To determine the exact sequence of bases two different technologies are available. The most frequently used technology is one developed by Professor Walter Gilbert. In this method the DNA is manipulated with an enzyme that breaks down a base or a combination of two bases. Lets take a piece of DNA with the following structure: A - T - C - A - T - C - G - G - T - A - A - T - A and lets expose it to an enzyme that breaks down C (cytosine). Than the result will be a mixture of the following pieces: A - T, A - T - C - G - G - T - A - A - T - A, A - T - C - A - T and G - G - T - A - A - T - A. Next we can apply the method of Southern, hence put the DNA on a gel and apply an electric voltage. The pieces will be sorted by size, the small A - T piece at the bottom and the big A - T - C - G - G - T - A - A - T - A on top. By using different combinations of enzymes on different places in the gel, it becomes possible to determine the precise sequences of the bases in the DNA. Because of this result this method is often referred to as sequencing.

Many things are possible with genetic manipulation. We have tried to explain this to you. Genetic manipulation has many advantages, but brings with it many disadvantages as well. This will be addressed in the next chapter.