What is Recombinant DNA?
Recombinant DNA is a man-made genetic code that incorporates genes from a organism merged with a vector and inserted into another organism, usually bacteria. This genetic line is generally something that would never occur in nature. As a form of genetic modification, it is used to identify the specific actions of proteins created by the gene. It can also be beneficial in the study of genetic disorders or the creation of organisms that have certain benefits over their natural structure, such as genetically-modified food.
The process was invented in the early 1970s at Stanford University, following the discovery of plasmids, the vector used in most recombinant DNA procedures. So what is the most common way to introduce recombinant DNA to bacteria?
Isolation of the desired gene is the first part of the process of creating recombinant DNA. Scientists need to determine what they would like to accomplish with the gene and how they will track the results. Depending on the study, different genes that can create specific proteins are chosen. This helps geneticists analyze the use of the gene as well as possibly create a different function within the chosen bacteria.
Using a restriction enzyme, the desired gene responsible for producing a particular protein in an organism is separated from the remaining chromosome. Restriction enzymes are naturally-occurring materials culled from bacteria and archaea, single-celled microorganisms with no nucleus or organelles. When these enzymes are introduced to the DNA, they can separate a nucleotide sequence from the rest of the structure. Essentially, this makes incisions in each strand of the DNA double helix, thereby the sugar-phosphate material is cut, releasing the gene for the recombinant process.
Preparing the Target DNA
Before the gene from the organism can be inserted into a receptive subject such as bacteria, certain processes must be accomplished. A portion of the bacteria’s DNA is removed. This is called a plasmid, a circular, double-stranded molecule within the DNA that is capable of replicating independently from the genomic DNA, commonly referred to as chromosomal DNA. Using the restriction enzymes again, a portion of the plasmid is removed and replaced with the desired gene. This gene is protected from further cutting using methylation, the addition of a hydrophobic alkyl molecule capable of repelling the restriction enzyme. A fusion enzyme must be used to link the new gene to the plasmid.
Insertion of the DNA into the Bacteria
The final process in what is the most common way to introduce recombinant DNA to bacteria is the insertion of the plasmid into the microorganism. This can be accomplished using one of two methods: chemical or electrical. Both the bacteria and the vector plasmid are placed together with a solution such as calcium chloride. By adjusting the temperature, usually lowering it to near freezing, it makes the bacteria more receptive to the absorption of the vector. The other way a plasmid can be inserted into the bacteria is through electroporation. This is much more reliable and can be controlled more efficiently by the geneticist. By applying a small pulse of electricity, it makes the bacteria susceptible to absorption through its pores.
"Bacterial Transformation" Plattsburgh State University of New York: https://faculty.plattsburgh.edu/donald.slish/Transformation.html
"The Basics of Recombinant DNA" Rensselaer Polytechnic Institute:https://www.rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/rdna/rdna.html
Recombinant formation of plasmids. (Supplied by Minestrone Soup at Wikimedia Commons; GNU Free Documentation License; https://upload.wikimedia.org/wikipedia/en/0/01/Recombinant_formation_of_plasmids.svg)