Gene Cloning Process
Gene cloning is the process of locating and copying a gene of interest out of DNA extracted from an organism. Gene cloning is a molecular technique in which a gene of interest is fused or incorporated into a self-replicating genetic element such as plasmid, which when introduced into a suitable host (usually bacteria), self-replicates and generates a large number of identical copies of the particular gene.
Basic steps are:
- Cut open the plasmid and “paste” in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA).
- Transform the plasmid into bacteria.
- Use antibiotic selection to identify the bacteria that took up the plasmid.
Cutting and pasting of DNA
- To be used in molecular cloning, both Insert DNA and Vector are treated with Restriction Enzymes.
- A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site.
- Many restriction enzymes produce cut ends with short, single-stranded overhangs.
- If two molecules have matching overhangs, they can base-pair and stick together.
- However, they won’t combine to form an unbroken DNA molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone.
The goal of the cloning is to insert a target gene into a plasmid. Fort this, using a carefully chosen restriction enzyme, digestion of plasmid and target gene fragment is done.
- The plasmid, which has a single cut site
- The target gene fragment, which has a cut site near each end
Then, the fragments are combined with DNA ligase, which links them to make a recombinant plasmid containing the gene.
There are two primary methods for transforming bacterial cells: heat shock and electroporation. In both cases, the bacterial cells have to be made competent or permeable to plasmids that you would like the cell to propagate.
- In order to prepare multiple copies of recombinant molecule, ligation mix can be introduced into bacteria, such as the harmless Escherichia coli used in labs, in a process called transformation.
- During transformation, specially prepared bacterial cells are given a shock (such as high temperature) that encourages them to take up foreign DNA.
- The cell-DNA mix is kept on ice (at 0˚C) and then exposed to 42˚C.
- Although heat shock transformation is a commonly used method in molecular biology, why a heat shock makes a bacterial cell take up the DNA is yet not actually answered.
- In general, it’s thought that the heat shock changes the fluidity of the membrane and/or causes pores (holes) to form, making it easier for the DNA to get across and enter the cell.
- For electroporation, the competent cells are placed on ice with the plasmid DNA. The plasmid-cell mixture is exposed to electrical current, opening pores in the cell membrane so that the plasmid can enter the cell.
- Some cells do not survive this electroporation treatment but many are able to replicate once medium is added. If the plasmid DNA solution has too much salt in it, arcing can occur, compromising the transformation.
- After transformation, cells are plated on a plate with growth supplemented with the selective antibiotics.
- A plasmid typically contains an antibiotic resistance gene, which allows bacteria to survive in the presence of a specific antibiotic.
- Thus, bacteria that took up the plasmid can be selected on nutrient plates containing the antibiotic.
- Bacteria without a plasmid will die, while bacteria carrying a plasmid can live and reproduce.
- Each surviving bacterium will give rise to a small, dot-like group, or colony (often termed as clone) of identical bacteria that all carry the same plasmid.
However, not all colonies will necessarily contain the right plasmid. That’s because, during a ligation, DNA fragments don’t always get “pasted” in exactly the way intended. Analysis of DNA from several colonies to observe whether each one contains the right plasmid should be done. Methods like restriction enzyme digestion and PCR are commonly used to check the plasmids.
During the process, it’s often possible for side products to form, in addition to the recombinant plasmid intended to build. For instance, while inserting a gene into a plasmid using a particular restriction enzyme, in some cases plasmid closes back up (without taking in the gene), and other cases the gene goes in backwards.
The selected clones are then individually picked, grown even further in a liquid medium and the DNA is extracted from them. As a part of the plasmid, this DNA fragment represents a quarter or more of the total DNA in the test tube and it could be effortlessly and endlessly amplified in E. coli. Thus through molecular cloning, a DNA fragment that represented a tiny fraction of cell genome is integrated into a bacterial plasmid and can be produced in large number (Mass production).
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