Molecular cloning reproduces the desired regions or fragments of a genome, enabling the manipulation and study of genes.
Describe the process of molecular cloning
- Cloning small fragments of a genome allows specific genes, their protein products, and non-coding regions to be studied in isolation.
- A plasmid, also known as a vector, is a small circular DNA molecule that replicates independently of the chromosomal DNA; it can be used to provide a “folder” in which to insert a desired DNA fragment.
- Recombinant DNA molecules are plasmids with foreign DNA inserted into them; they are created artificially as they do not occur in nature.
- Bacteria and yeast naturally produce clones of themselves when they replicate asexually through cellular cloning.
- recombinant DNA: DNA that has been engineered by splicing together fragments of DNA from multiple species and introduced into the cells of a host
- molecular cloning: a biological method that creates many identical DNA molecules and directs their replication within a host organism
- plasmid: a circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa
In general, the word “cloning” means the creation of a perfect replica; however, in biology, the re-creation of a whole organism is referred to as “reproductive cloning.” Long before attempts were made to clone an entire organism, researchers learned how to reproduce desired regions or fragments of the genome, a process that is referred to as molecular cloning.
Cloning small fragments of the genome allows for the manipulation and study of specific genes (and their protein products) or noncoding regions in isolation. A plasmid (also called a vector) is a small circular DNA molecule that replicates independently of the chromosomal DNA. In cloning, the plasmid molecules can be used to provide a “folder” in which to insert a desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, the fragment of DNA from the human genome (or the genome of another organism that is being studied) is referred to as foreign DNA (or a transgene) to differentiate it from the DNA of the bacterium, which is called the host DNA.
Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism such as antibiotic resistance (the ability to be unaffected by antibiotics). Plasmids have been repurposed and engineered as vectors for molecular cloning and the large-scale production of important reagents such as insulin and human growth hormone. An important feature of plasmid vectors is the ease with which a foreign DNA fragment can be introduced via the multiple cloning site (MCS). The MCS is a short DNA sequence containing multiple sites that can be cut with different commonly-available restriction endonucleases. Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, these are called “sticky ends.” Addition of an enzyme called DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, any DNA fragment generated by restriction endonuclease cleavage can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction endonuclease.
Molecular Cloning: This diagram shows the steps involved in molecular cloning, where regions or fragments of a genome are reproduced to allow the study or manipulation of genes and their protein products.
Recombinant DNA Molecules
Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they are created artificially and do not occur in nature. They are also called chimeric molecules because the origin of different parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis. Proteins that are expressed from recombinant DNA molecules are called recombinant proteins. Not all recombinant plasmids are capable of expressing genes. The recombinant DNA may need to be moved into a different vector (or host) that is better designed for gene expression. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors so that scientists can control the expression of the recombinant proteins.
Unicellular organisms, such as bacteria and yeast, naturally produce clones of themselves when they replicate asexually by binary fission; this is known as cellular cloning. The nuclear DNA duplicates by the process of mitosis, which creates an exact replica of the genetic material.