Genetic engineering: principles and techniques

In a nutshell

Genetic engineering involves taking a gene from one organism and inserting it into another organism using a vector. There are certain stages that must be carried out; from isolating the target gene to the transport of the gene into the host cell.

Genetic engineering

Definition

Genetic engineering, also known as recombinant DNA technology and genetic modification, is the process of taking a gene from one organism and inserting it into another using a vector. A vector is used to transport a segment of DNA into a host cell.

There are three main stages that must be carried out during genetic engineering.

1.	Obtaining the target gene.
2.	Inserting the gene into a vector.
3.	Getting the vector to transport the gene to the host cell.

Obtaining the target gene

The target gene is isolated using an enzyme called reverse transcriptase. It catalyses the formation of complementary DNA (cDNA) from mRNA. Primers and DNA polymerase will be added to turn the cDNA into double stranded DNA that encodes the target gene. The polymerase chain reaction (PCR) can then be used to amplify the gene.

Alternatively, a DNA probe can locate the target gene and restriction enzymes can be used to cut it out. This will often leave sticky ends which are short chains of unpaired bases.

Inserting the gene into a vector

A common vector used in genetic engineering is plasmids. These are found in abundance in bacteria and they can be manipulated to carry the target gene. The plasmid is mixed with the same restriction enzyme that was used to obtain the target gene. This will create sticky ends in the vectors that are complementary to the sticky ends created in the target gene.

Note: Bacteriophages are viruses that infect bacteria and they can also be used as vectors.

The vector and the gene are mixed together with the enzyme DNA ligase which catalyses the ligation reaction. This joins the sugar-phosphate backbones of the two fragments.

Biology; Manipulating genomes; KS5 Year 12; Genetic engineering: principles and techniques — 1. DNA from the target cell, 2. Bacterial plasmid, 3. Antibiotic resistance marker, 4. Second antibiotic resistance marker, 5. Target DNA and the plasmid are cut using the same restriction endonuclease, 6. Sticky ends, 7. The DNA fragment and plasmid are mixed with DNA ligase, 8. Plasmid with target gene inserted.

Target gene transport

The plasmid must then be put into a bacterial cell. It is difficult for DNA to cross the plasma membrane so there are a few methods that can be used to help this process.

Electroporation	Bacterial cells and the plasmid are placed into an electroporator. Pulses of high voltage are applied to the bacterial cell that disrupt the membrane and allow the bacterial cell to take up the plasmid.
Heat shock treatment	In the presence of calcium chloride, bacterial cells are subjected to periods of hot ( $42\ \degree C$ ) and cold ( $0\ \degree C$ ). This increases the permeability of the bacterial membrane and allows the uptake of the plasmid.