=Gene Cloning= Prior to the discovery of PCR, genetic sequences had to be replicated in vivo (within a living organism). The idea was to insert the target sequence (usually a gene) into a bacterial genome, so that as the bacteria replicated they would also copy the DNA sequence within them. Bacteria were chosen primarily because they replicate quickly (every 20mins in optimal conditions). Bacteria have a single large circular piece of chromosomal DNA as well as smaller circular pieces of DNA known as Plasmids. These plasmids were an attractive target as they are capable of replicating independently of the chromosomal DNA. ===Bacterial Plasmids=== [image:] Plasmids normally allow bacteria to undergo a process known as conjugation. Bacterial Conjugation is the transfer of genetic material (usually the plasmid) between bacterial through direct cell-to-cell contact (this is often incorrectly referred to as bacterial sex). Some bacteria are capable of taking up exogenous (foreign) DNA from their environment and incorporating it into their genome. This process is known as bacterial transformation. Foreign plasmids can also be taken up and incorporated as part of the bacterial genome. ==Steps in Gene Cloning:== [image:] # The target gene is isolated and cut out using a ‘sticky end cutter’ restriction enzyme. # The bacterial plasmid is cut open using the same restriction enzyme. # DNA ligase ‘sticks’ (ligates) the target gene into the plasmid. The resulting plasmid is known as a recombinant plasmid. Next the recombinant plasmid is inserted into the bacterium by a process known as transformation. # The bacteria are then placed in a growth reactor, providing the optimal growth conditions, allowing the bacteria to replicate many times. Finally the bacteria are lysed (cut open) and the plasmids are extracted. Reporter Genes: Plasmids containing antibiotic resistance genes are commonly used. These genes act as a selectable marker, making it easy to identify (and eliminate) those bacteria that did not take up the plasmid.
Credit: Ben Himme