Genetic Engineering: Techniques, Vectors, and PCR Explained
Genetic Engineering: An Overview
Genetic Engineering is a technique that consists of introducing genes into the genome of an individual who lacks them. It is performed by restriction enzymes that are able to “cut” DNA at specific points. Recombinant DNA is formed by inserting a DNA segment into a foreign DNA receiver. For example, the introduction of viral DNA into a cell’s DNA.
DNA Recombination Techniques
This includes a set of biotechnology techniques. DNA recombination allows us to obtain unlimited quantities of DNA fragments, which also carry the desired gene or genes. This DNA can be incorporated into the cells of other organisms where it can “express” the information of these genes. If such is the gene which regulates the production of insulin, when introduced, it would “force” a bacteria to manufacture insulin.
Basic Steps in Recombinant DNA Technology
In recombinant DNA technology, four basic steps can be distinguished:
- Specific DNA Cutting: Cutting DNA into small and manageable pieces through the use of a type of enzyme known as restriction enzymes, which can be considered as “molecular scissors.” These enzymes were isolated and identified in bacteria with different names, being characteristic of these two principles:
- Each restriction enzyme recognizes a specific sequence of nucleotides at that point and cuts each of the DNA strands.
- The free ends remaining are called sticky ends because they can join other DNA fragments that have been cut with the same restriction enzyme.
- Insertion of DNA Fragments: This insertion is done in cloning vectors, which are carrier agents able to introduce them into host cells. Cloning vectors are small DNA molecules that have the capacity to self-replicate inside host cells. Three types of cloning vectors are frequently used: plasmids, viruses, and cosmids.
Types of Cloning Vectors
- Plasmids: They are small and circular duplex DNA molecules that are found in bacteria. They are not part of the bacterial chromosome and are capable of independent replication, and they have their own origin of replication. In addition, many plasmids carry genes that confer resistance to some antibiotics, which gives the host a feature that allows it to easily identify the gene carrier.
- Virus Phage: These are viruses that infect bacteria. Their use as vectors is because during transduction, some genes can be incorporated into the host’s genome. When the phage infects another bacterium, the genes may transfer from the first. The most employed are bacteriophages lambda, which are capable of carrying much plasmid DNA.
- Cosmids: These are hybrid vectors between phage and plasmid. Their DNA can replicate in a cell as a plasmid or be packaged as a phage. They are so called because they carry the bacteriophage λ cos sequence, which includes the genes necessary for the genetic material to be packed. Furthermore, they have a plasmid origin of replication, one or more marker genes that confer resistance to certain antibiotics, and a number of restriction sites where you can insert the foreign DNA. The advantage of cosmids is that the proportion of foreign DNA they can carry may be much greater.
PCR (Polymerase Chain Reaction)
Requires a DNA sample, nucleotide sequence, heat source, DNA polymerase, and an appropriate amount of nucleotides.
- Denaturation: The heat denatures the DNA, and each of the single strands serves as a template to synthesize a complementary strand.
- Hybridization: Cool the reaction mixture to allow the primers to join by hydrogen bonds to each of the ends of the DNA strands that have been separated in the previous stage.
- Extension: New DNA strands are created by the action of Taq polymerase, adding nucleotides to the 3′ end of the primer.
Result: 2 DNA molecules become 4, and later, 8.