What is Recombinant DNA Technology?

What is Recombinant DNA Technology?

Recombinant DNA technology is a form of genetic engineering that combines DNA from two different organisms. The result is a genetically modified organisms that can produce a target protein or product. This has definitely affected various industries in biotechnology, from medicine to agriculture.

General Steps to make a Recombinant Organism

1. Isolation of DNA

This step involves extraction of DNA from an organism using mechanical or chemical factors, such as detergents to dissolve cell membranes. At this point, the donor cell(s) are disrupted, and now the DNA needs to be precipitated. This is usually done with ethanol and salt, which lowers the solubility of the DNA and causes precipitation, which can be seen as a white stringy mass. The DNA is then spun down in a centrifuge. This process isolates both the vector (usually a plasmid) and the gene of interest.

2. Restriction Enzymes and Ligation

The next step after the DNA has been centrifuged is to cleave both the vector and gene of interest with the same restriction enzyme. There is no "one size fits all" enzyme, rather there are many types of enzymes that cut at specific DNA sequences. Restriction enzymes open the plasmid vector so that the gene of interest can fit inside. After enzymatic digestion, the result are sticky ends or cohesive plasmid and gene of interest ends. Cohesive ends of both the vector and gene of interest is essential for proper ligation. This is done using DNA ligase, which joins the open vector with the gene of interest, forming a recombinant plasmid.

3. Transformation of host cells

After recombinant plasmids have been made, the next step is to introduce the new plasmids into a host. This host usually is a non-pathogenic E. Coli strain or a species of Yeast. However, these plasmids have to be forcefully introduced into the host, as some are not competent.

• Calcium chloride treatment and heat shock to make a leaky membrane (bacteria)

• Electroporation (Both)

• Lithium Acetate (Yeast)

• Heat shock (Both)