Molecular Cloning

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 (see the figure below).

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.

Art Connection

Figure illustrates the steps in molecular cloning into a plasmid called a cloning vector. The vector has a lacZ gene, which is necessary for metabolizing lactose, and a gene for ampicillin resistance. Within the lacZ gene are restriction sites, sequences of DNA cut by a particular restriction enzyme. The DNA 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 DNA, such that each strand has an overhanging single-stranded bit of DNA. On one strand, the sequence of the overhang is GATC, and on the other, the sequence is CTAG. These two sequences are complementary, and allow the fragment of foreign DNA 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 lacZ 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 lacZ gene and produce white colonies. Thus, colonies containing the cloned DNA can be selected on the basis of color.

This diagram shows the steps involved in molecular cloning.

You are working in a molecular biology lab and, unbeknownst to you, your lab partner left the foreign genomic DNA that you are planning to clone on the lab bench overnight instead of storing it in the freezer. As a result, it was degraded by nucleases, but still used in the experiment. The plasmid, on the other hand, is fine. What results would you expect from your molecular cloning experiment?

There will be no colonies on the bacterial plate.
There will be blue colonies only.
There will be blue and white colonies.
The will be white colonies only.

Answer

The experiment would result in blue colonies only.

View an animation of recombination in cloning from the DNA Learning Center in the video below:

Molecular Cloning