Genetic Advances
4. Recombinant DNA
Recombinant DNA
Recombinant DNA is the combination of DNA from two different sources. It is useful in gene cloning and in identifying the function of a gene, as well as producing useful proteins. Human insulin for treating diabetes has been produced through recombinant DNA methods. In this process, a gene of interest (or piece of DNA of interest) is placed into a host cell, such as a bacterium, so the gene can be copied (and cloned) and the protein that results from that gene can be produced.
To place the gene of interest into a host cell, a vector, or carrier molecule, is needed to the carry foreign DNA into the host cell. Bacteria have small accessory rings of DNA in the cytoplasm, called plasmids. When putting foreign DNA into a bacterium (a host cell), the plasmids are often used as a vector. Viruses can also be used as vectors.
The first step of making recombinant DNA involves a restriction enzyme that cuts the vector and the foreign (exogenous) DNA. Restriction enzymes cut DNA at specific sequences, such as GAATTC as shown in Figure below . There are more than 3,000 known restriction enzymes, most cutting the DNA at a unique sequence. This reaction results in the plasmid opening up a gap with “sticky ends,” which can attach with the complimentary base pairs on the sticky ends of the foreign DNA. Then the enzyme DNA ligase seals the foreign DNA in its new place inside the plasmid. These altered plasmids are introduced back into the bacteria, a process called transformation (Figure below). The bacteria will express the foreign gene.
Restriction enzymes cut DNA at specific sequences, in this example the sequence GAATTC." The enzyme cuts between the G and A on each strand, producing overhanging sticky ends.
This image shows a line drawing of a plasmid. The plasmid is drawn as two concentric circles that are very close together, with two large segments and one small segment depicted. The two large segments (1 and 2) indicate antibiotic resistances usually used in a screening procedure, and the small segment (3) indicates an origin of replication. The resulting DNA is a recombinant DNA molecule.
One application of recombinant DNA technology is producing the protein insulin, which is needed to treat diabetes. Previously, insulin had been extracted from the pancreases of animals. Through recombinant DNA technology, bacteria were created that carry the human gene which codes for the production of insulin. These bacteria become tiny factories that produce this protein. A step-by-step depiction of the cloning of the insulin gene is shown below in (Figure below).
A step-by-step depiction of the cloning of the insulin gene. The plasmid is opened up with restriction enzymes and the gene of interest (human cDNA) is inserted into the plasmid with complementary linkers, producing the recombinant plasmid. The plasmid is transfected into bacterial cells, where the human protein is produced.
CK-12 Foundation, Biology. http://creativecommons.org/licenses/by-nc-sa/3.0/