Biomolecular Engineering - Biomolecular Engineering of Molecules - Recombinant DNA - Method


The traditional method for creating recombinant DNA typically involves the use of plasmids in the host bacteria. The plasmid contains a genetic sequence corresponding to the recognition site of a restriction endonuclease, such as EcoR1. After foreign DNA fragments, which have also been cut with the same restriction endonuclease, have been inserted into host cell, the restriction endonuclease gene is expressed by applying heat, or by introducing a biomolecule, such as arabinose. Upon expression, the enzyme will cleave the plasmid at its corresponding recognition site creating sticky ends on the plasmid. Ligases then joins the sticky ends to the corresponding sticky ends of the foreign DNA fragments creating a recombinant DNA plasmid.

Advances in genetic engineering have made the modification of genes in microbes quite efficient allowing constructs to be made in about a weeks worth of time. It has also made it possible to modify the organism's genome itself. Specifically, use of the genes from the bacteriophage lambda are used in recombination. This mechanism, known as recombineering, utilizes the three proteins Exo, Beta, and Gam, which are created by the genes exo, bet, and gam respectively. Exo is a double stranded DNA exonuclease with 5’ to 3’ activity. It cuts the double stranded DNA leaving 3’ overhangs. Beta is a protein that binds to single stranded DNA and assists homologous recombination by promoting annealing between the homology regions of the inserted DNA and the chromosomal DNA. Gam functions to protect the DNA insert from being destroyed by native nucleases within the cell.

Read more about this topic:  Biomolecular Engineering, Biomolecular Engineering of Molecules, Recombinant DNA

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