The Molecular Cloning


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This figure illustrates the steps in molecular cloning into a plasmid called a cloning vector. The vector has a lac Z gene, which is necessary for metabolizing lactose, and a gene for ampicillin resistance. Within the lac Z gene are restriction sites, sequences of D N A cut by a particular restriction enzyme. The D N A to be cloned and the plasmid are both cut by the same restriction enzyme. The restriction enzyme staggers the cuts on the two strands of D N A, such that each strand has an overhanging single-stranded bit of D N A. On one strand, the sequence of the overhang is G A T C, and on the other, the sequence is C T A G. These two sequences are complementary, and allow the fragment of foreign D N A to anneal with the plasmid. An enzyme called ligase joins the two pieces together. The ligated plasmid is then transformed into a bacterial strain that lacks the lac Z gene and is sensitive to the antibiotic ampicillin. The bacteria are plated on media containing ampicillin, so that only bacteria that have taking up the plasmid; which has an ampicillin resistance gene; will grow. The media also contains X gal, a chemical that is metabolized in the same way as lactose. Plasmids lacking the insert are able to metabolize X gal, releasing a dye from X gal that turns the colony blue. Plasmids with the insert have a disrupted lac Z gene and produce white colonies. Thus, colonies containing the cloned D N A can be selected on the basis of color.
This diagram shows the steps involved in molecular cloning. Source: OpenStax Biology 2e

OpenStax Biology 2e

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 genome fragments allows researchers to manipulate and study specific genes (and their protein products), or noncoding regions in isolation. A plasmid, or vector, is a small circular DNA molecule that replicates independently of the chromosomal DNA. In cloning, scientists can use the plasmid molecules 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, scientists call the DNA fragment from the human genome (or the genome of another studied organism) foreign DNA, or a transgene, to differentiate it from the bacterium’s DNA, or 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). Scientists have repurposed and engineered plasmids 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 scientists can introduce a foreign DNA fragment via the multiple cloning site (MCS). The MCS is a short DNA sequence containing multiple sites that different commonly available restriction endonucleases can cut. 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 DNA strands, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, we call them “sticky ends.” Adding the enzyme DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, scientists can splice any DNA fragment generated by restriction endonuclease cleavage between the plasmid DNA’s two ends that has been cut with the same restriction endonuclease.

Source:

Clark, M., Douglas, M., Choi, J. Biology 2e. Houston, Texas: OpenStax. Access for free at: https://openstax.org/details/books/biology-2e