Genetic Engineering: Fundamentals of Genetic Engineering

Fundamentals of Genetic Engineering

Genetic engineering is the process of transferring specific genes from the chromosome of one organism and transplanting them into the chromosome of another organism in such a way that they become a reproductive part of the new organism. The process that produces the resulting recombinant DNA involves four steps:

1. The desired DNA is cleaved from the donating chromosome by the action of restriction enzymes, which recognize and cut specific nucleotide segments, leaving a “sticky end” on both ends. The restriction enzymes also splice the receiving chromosome in a complementary location, again leaving “sticky ends” to receive the desired DNA.
2. The desired DNA fragment is inserted into a vector, usually a plasmid, for transfer to the receiving chromosome. Plasmids are an ideal vector because they replicate easily inside host bacteria and readily accept and transfer new genes. Plasmids are circular DNA molecules found in the cytoplasm of bacteria that bond with the desired DNA fragment with the help of the joining enzyme, DNA ligase, to create the resulting recombinant DNA.
3. When the host cell reproduces, the plasmids inside also reproduce, making multiple clones of their DNA. Because the plasmid DNA contains the desired as well as unwanted DNA clones, the entire product is referred to as a gene library. The desired gene is similar to one book in that library.
4. To recover the desired DNA, the current technology is to screen unwanted cells from the mixture and then use gel electrophoresis to separate the remaining genes by movement on an electric grid. Gel electrophoresis uses a positively charged grid to attract the negatively charged DNA fragments, thereby separating them by size, because the smaller ones will migrate the most. Radioactive or fluorescent probes are added, which attract and bind with the desired DNA to produce visible bands. Once isolated, the DNA is available for commercial use.

In 1973, researchers Cohen and Boyer created an interesting model for screening the host cells to finds the desired DNA fragment. In their experiment, they inserted the desired DNA and a DNA segment that made the host bacteria resistant to a particular antibiotic, tetracycline. When the antibiotic was applied to the general population, only those bacteria that had received the plasmid survived—so they knew their desired DNA fragment was located in the surviving bacteria.