Preparatory work on genetic engineering
When the researchers realized that the cells of all living things on earth, from the most primitive microorganisms to humans, use the same code, one idea was very obvious: it should be possible to exchange genetic information between different organisms. This happens in nature anyway, as the American Barbara McClintock (1902–1992) recognized: Her experiments proved that pieces of the genetic material jump back and forth between individual maize plants. Today these jumping genes, which occur in many organisms, are called transposons.
But how can genetic information be transferred from one organism to another in the laboratory? Salvador Luria (1912–1991) carried out preparatory work for such genetic engineering by examining so-called bacteriophages, viruses that only attack bacteria. Certain bacteriophages grow well on some strains of bacteria, but poorly on others. Obviously, some bacteria prevented the virus infection with a certain mechanism that Luria called restriction.
This restriction works as follows: The bacteria have enzymes that recognize certain sections of the DNA of bacteriophages and then cut them up. This way the virus can no longer multiply. The bacterium itself protects its DNA with a small chemical reaction called methylation. There are three types of these enzymes; With the help of type II it was finally possible to transfer genetic material from one organism to the other. Note: according to ABBREVIATIONFINDER.ORG, DNA stands for Deoxyribonucleic Acid.
How genetic engineering works
Many of these type II restriction enzymes recognize so-called palindromic sections in the genome, in which a short sequence of nucleotides on one DNA strand is repeated in the other DNA strand in the opposite direction. The enzyme with the name EcoRI, for example, attaches itself to the GAATTC sequence of one strand and cuts it between the G and the first A. The other DNA strand of the double helix naturally has the same sequence GAATTC at this point in the opposite direction, the EcoRI also between G and A cuts up. This creates two pieces of DNA from which a single strand, four nucleotides long, with the sequence TTAA protrudes (G and C are still related).
Genetic engineers call such protruding ends sticky because they are particularly easy to connect to a piece of DNA that was also cut with EcoRI – after all, exactly the right counterpart protrudes from it. In principle, a piece of DNA can be cut out of the genome of an organism with such a restriction enzyme that makes sticky ends. This is glued into the genome of another organism that has been cut open with the same restriction enzyme.
In this way, for example, a piece of DNA from a rabbit can be glued into the DNA of a virus and then transferred into a monkey cell. Just as it did before in rabbits, this gene also functions later in monkeys.
Sequences – the Sanger method
Over the years, new methods have been developed with the help of which the genetic material can be better analyzed and also better transferred to other organisms. Above all, it was important to be able to determine the sequence of the genetic information. The biochemist Frederick Sanger (1918-2013) used the following method.
When DNA is heated, the double strands separate from each other. The four nucleotides A, C, G and T, which are necessary for an addition to the double strand, and an enzyme that neatly incorporates these nucleotides are added to these single strands in four different reaction vessels. In addition, a small amount of a slightly modified nucleotide A that no longer has a docking point for the next nucleotide is placed in one of the vessels. As soon as such a modified nucleotide A is incorporated into the newly formed strand, the new DNA strand ends at this point. However, since there are also a lot of intact A-nucleotides present, DNA fragments of different lengths are created, all of which end with an A. Now you can use gel electrophoresis (a method of separating molecules) to determine how long each of these fragments is, and deduce from this at which point in the DNA there is an A. The same method is repeated in the three other reaction vessels, in which either a modified C, G or T is added.
Initially, scientists used this so-called Sanger method to determine the sequence of relatively short pieces of DNA with around 1000 nucleotides. It seemed hopeless to determine the entire sequence of the approximately 78 billion nucleotide pairs in a lung fish or even the approximately 3 billion nucleotide pairs in a person. Only when the Sanger method was automated was it possible to analyze the genetic makeup of such organisms largely completely with the help of computers.
Another breakthrough came with the so-called polymerase chain reaction (PCR). This method of duplicating the genetic material in the DNA works similarly to Sanger sequencing, but without the modified nucleotides that break off a newly formed DNA strand: If you put a piece of single DNA strand in a reaction vessel, it becomes a complete one Double strand added. This is separated again into individual strands, whereupon it is supplemented again. Repeated 30 times, this procedure produces over 1 billion copies from one piece of DNA. So the PCR turns a small amount of a piece of genetic material into large amounts. This is exactly the method that paleontologists use today to analyze the genetic material of extinct animals, which is only present in fragments.