Free Course Sample
This sample represents a section of the Biotechnology/Pharmacogenetics course. Biotechnology and Pharmacogenetics are listed as subject areas to be reviewed in the evaluating exam syllabus posted on PEBC website.
A clone is a genetically identical copy of a plant, animal, microorganism or gene; both the original and the clone are derived from a single common ancestor gene, cell or organism. Identical twins are natural clones from one fertilized egg. A gene is said to be cloned when its sequence is multiplied many times in a common laboratory procedure.
The cloning of a gene is a step in determining its sequence and initiating many types of experiments to understand its function and biology. Cloning of animals and plants can be the basis of developing more efficient methods to produce superior breeds of animals. Cloning also enables genes to be added (such as those for human proteins) to produce animals that can produce hormones and pharmaceuticals in milk, eggs or other products. Transferring single genes between different animals, turning existing genes on or off, or removing a gene from its original position and placing it in a new position in the same organism are all cloning techniques. Animals or microbes that have a new gene inserted into them are called GMOs or transgenics.
Cloning of DNA from any organism involves five general steps (Figure 1):
Step 1 Cut DNA at precise locations to yield the DNA fragment of interest. This task is performed by restriction endonucleases, which are DNA-cutting enzymes found in bacteria and harvested from them for use. These restriction endonucleases recognize and cleave DNA at specific DNA sites. Some restriction endonucleases such as BamHI and HindIII make staggered cuts on the two DNA strands, leaving two to four nucleotides of one strand unpaired at each resulting end. These unpaired strands are called “sticky ends” because they can base pair with a complementary sequence. Other restriction endonucleases such as HaeIII and PvuII cleave both DNA strands at the opposing bonds, leaving no unpaired bases on the ends; these are often called “blunt ends”.
Step 2 Select a cloning vector. A cloning vector is a small piece of DNA with self-replication capability. Cloning vectors, which act as delivery agents, include plasmids, bacteriophages, yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs). Plasmids are circular DNA that replicate separately from the host DNA. They usually carry few genes and have a single origin of replication. Plasmids are replicated by the same machinery that replicates BAC. Some plasmids are copied at about the same rate as the chromosome, so a single cell is apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate and a single cell may have 50 or more of them.
Step 3 Link the DNA fragment to be cloned to the cloning vector to yield a recombinant DNA. Recombinant DNA, the general name for taking a piece of one strand of DNA and combining it with another strand is sometimes referred to as a “chimera”. By combining two or more different strands of DNA by using the enzyme DNA ligase scientists are able to create a new strand of DNA. The most common recombinant process involves combining DNA of two different organisms. The ability to produce recombinant DNA molecules has not only revolutionized the study of genetics, but has laid the foundation for much of the biotechnology industry. The availability of human insulin for people with diabetes, human factor VIII for males with hemophilia, and other proteins used in human therapy were all made possible by recombinant DNA.
Step 4 Insert the recombinant DNA into a host cell which will provide the enzymatic machinery for DNA replication. When plasmids are used as cloning vectors, they can be introduced into bacteria by a process called transformation. Transformation can be achieved by temperature shock, which refers to rapidly shifting the temperature from 0°C to 37°C. An alternate method is electroporation, where cells are subjected to high voltages. Non-bacterial transformations can be achieved by microinjection where the DNA is injected directly into the nucleus of the cell being transformed. In 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 is the process of transfection, which is almost equivalent to transformation, except that a phage is used instead of bacteria. The process involves the use of lambda or MI3 phages to produce phage plaques that contain recombinant DNA. The recombinants that are created can be identified by using various selection methods described in Step 5.
Step 5 Select host cells that contain the recombinant DNA by one of the following methods:
Antibiotic resistance selection: The plasmid can be designed to carry an antibiotic resistance gene such as ampicillin or tetracycline. Once transformed bacteria cells are exposed to the appropriate antibiotic, only cells carrying the recombinant DNA (plasmid) survive.
DNA hybridization is the most common sequence-based process for detecting a particular gene. This technique makes use of labeled DNA or ribonucleic acid (RNA), known as probes, complementary to the DNA of interest. Transformed bacteria colonies are lysed on a piece of nitrocellulose paper; then the probe is added. Following incubation and washing, the labeled DNA is detected bound to the bacteria cells carrying the recombinant DNA.
Other selection markers can be for color changes or any other characteristic that can distinguish transformed cells from untransformed cells
Figure 1: The process of DNA Cloning