Recombinant DNA technology has been popularized as genetic engineering, so we will explain them as one.

In the process of Recombinant DNA technology, DNA must be "fractured" to give fragments containing genes. Plasmid "vectors" carry the clone DNA sequence. Host cells are grown in order to allow the cloned sequence to grow and amplify. This is the method for identifying the presence of a cloned sequence.

A Recombinant DNA is created by forming a plasmid vector which is digested with EcoRI at a single site to produce two sticky ends. A sample of human DNA is also digested with EcoRI to produce pieces with the same sticky ends. These DNA's are copied from messenger RNA using reverse transcriptase from a retrovirus. The two samples are then mixed and allowed to hybridize, some molecules will form with pieces of human DNA inserted into the plasmid vector at the EcoRI site. DNA ligase is used to covalently link the fragments.

There are three different ways of inserting foreign DNA into host cells.

1. Transformation- can contact and take up isolated DNA from other bacteria.

2. Transfection- the uptake, incorporation, and expression of foreign DNA in which the cell walls are removed before DNA can enter the cell.

3. Electroporation- cells are exposed to rapid pulses of high-voltage current, which temporarily renders the plasma membrane permeable to macromolecules in the medium.

During the process of insertional mutagenesis, the plasmid vector contains another identifible gene, with the coding sequence of this gene containing the restriction site for insertion. Insertion of the foreign DNA at this site interrupts the reading frame of the gene and results in insertional mutagenesis.

A probe is used to detect the desired DNA during the audioradiography. Audioradiography is "the detection of a radioactive substance in a cell or organism by putting it in contact with a photographic emulsion and allowing the material to "take its own picture." The emulsion is developed, and the location of the radioactivity in the cell is seen by the presence of silver grains in the emulsion" (Purves, Orians, and Heller, G4).

This information was taken from Fall 1996, Biology 181 sections 1-4, Lecture Notes of Richard B. Hallick and William J. Grimes.