Recombinant DNA involves taking a piece of DNA and combining it with another strand of DNA.
There are three methods of doing this: Transformation, Non-Bacterial Transformation and Phase Introduction.
Transformation
- Select a piece of DNA to be inserted into a vector (Plasmid).
- Cut the selected DNA using a restriction enzyme and then insert the DNA into the vector with DNA Ligase.
- Add markers to the insert.
- Insert the vector into a host cell e.g. E. Coli.
Non-Bacterial Transformation
Same as above except that a bacterial host is not used (Perhaps eukaryotic cell?).
Typically the DNA is inserted in one of two ways:
Microinjection - DNA is inserted directly into the nucleus of the cell being transformed.
Biolistics - The host cells are bombarded with high velocity microprojectiles, such as particles of gold or tungsten that have been coated with DNA.
Phage Introduction
The process of 'transfection' which is equivalent to transformation except a phage (virus that infects bacteria) is used instead of bacteria.
How rDNA works
The host cell expressed protein from the recombinant genes. A significant amount of recombinant protein will not be produced by the host unless expression factors are added. Protein expression depends upon the gene being surrounded by a collection of signals which provide instructions for the expression and translation of the gene by the cell. These signals include the promoter, the ribosome binding site and the terminator. Expression vectors contain these signals. Signals are species specific e.g. E. Coli signals must be used with E.Coli.
Problems occur if the gene contains introns or signals which act as signals to the bacterial host. This results in premature termination and the protein will be severely affected.
Source: http://rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/rdna/rdna.html
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