DNA cloning is a molecular biology technique that makes many identical copies of a gene. The cloned gene is then inserted into a circular piece of DNA called a plasmid, which can be infected with bacteria that will carry the gene and make protein.
The cloning experiment by Campbell and Wilmut was a breakthrough because it demonstrated that it could be possible to clone mammals using nuclei from mature cells instead of embryonic ones.
1. Isolate the Gene
A gene is an exact copy of a single sequence of nucleotides in a cell’s DNA. Cloning is the process of isolating and making these exact copies into a larger, more easily manipulated piece of DNA called a plasmid. This makes it possible to study the function of a particular gene in the laboratory.
Many cloning experiments begin by creating or obtaining a library of cDNA containing the interesting DNA sequence. This library can then be screened by a variety of techniques to isolate a clone that contains the desired DNA sequence.
For example, if an experimentalist is interested in the gene that encodes for a protein produced by erythrocytes (red blood cells), they may want to create a cDNA library from erythrocytes. This library would be highly enriched with cDNA molecules that encode for the protein of interest, thus greatly reducing the amount of screening necessary to find the correct molecule.
Other ways of isolating cDNA are by using plus/minus screening or differential display. These approaches work by comparing populations of cells treated with either growth factors like NGF or PDGF or hormones like estrogen to control the expression of different genes in each population. By screening populations of cells that have been exposed to control and then to the experimental manipulation, the experimentalist hopes to identify cDNA molecules that are unique in their expression pattern.
2. Extract the DNA
Students use a variety of laboratory techniques to extract DNA. DNA is a long chain of nucleotides, and it is difficult to separate from other macromolecules, such as proteins. Once isolated, the DNA is ready for cloning.
Molecular cloning involves inserting foreign DNA into the genome of a host organism. A gene-containing DNA sequence is extracted from a living cell and cut by restriction enzymes at specific sites to begin the process. The ends of the DNA are then joined together using a special enzyme called DNA ligase. The ligated DNA is called a plasmid.
The plasmid can be pasted into a bacterium, which will then make many copies of the plasmid over time. If the plasmid contains a gene that produces a protein, it can be expressed in large quantities in the host bacterium. This can be very useful in the production of new drugs and biofuels.
A plasmid is also easier to handle and transform than linear DNA. It is easier to shear and has fewer overlapping restriction enzyme sites. Moreover, it is often accompanied by a promoter region that controls the expression of the gene.
This six- to eight-week exercise combines wet lab experiences, such as DNA extraction, polymerase chain reaction, PCR amplification, and cloning, with bioinformatics tasks such as Basic Local Alignment Search Tool searches, contig construction, intron identification, and six-frame translation. The goal of this project is for the instructor and students to be listed as coauthors on a GAPDH gene sequence published in the National Center for Biotechnology Information GenBank. Instructors can obtain the necessary materials for this project by purchasing a Cloning and Sequencing Explorer Series kit from Bio-Rad Laboratories (catalog no. 166-5000EDU).
3. Make a plasmid
Before cloning an entire organism, scientists first learn to copy and reproduce short stretches of DNA. This is called molecular cloning. It’s also what allows us to make insulin in bacteria, for example.
To do this, scientists take a circular piece of plasmid DNA and “paste in” the gene of interest from another species. Both the plasmid and the gene have DNA sequences with matching overhangs at their ends, so they’re compatible with each other. The plasmid and the gene fragment are then incubated with an enzyme (DNA ligase) that covalently joins their complementary ends, producing a single recombinant DNA molecule.
The recombinant plasmid is then introduced into bacteria, which take up the plasmid and reproduce it, making more recombinant DNA molecules. The recombinant DNA can then be used for further experiments, for instance, to see what protein it codes for.
Scientists use specialized vectors containing the necessary transcription and translation signals in different host organisms. These are known as expression vectors.
Cloning is a huge area of research, and new techniques are always being developed. Some of these may even allow scientists to clone an entire human.
While cloning humans may seem like a distant goal, it’s important to remember that human cells already contain many overlapping pieces of DNA, so we share many genes with other people. Even so, there are lots of differences between people, and it’s also impossible to clone an entire human being without disrupting the rest of the cell and possibly the individual’s personality. Despite these limitations, scientists are working on this issue, and the results may be astounding. Someday, cloning could allow scientists to grow healthy replacement organs for patients with damaged or defective ones.
4. Create an embryo
A clone is an exact copy of something, including genes, cells, animals, and plants. Cloned cells are used in medical research and gene therapy. Clones are also useful for teaching about how genes function in cells. Scientists create clones in labs by using a process called artificial embryo twinning. This technique mimics the natural process that forms twins in the first days after an egg and sperm join. The two resulting embryos have the same DNA because they come from the same fertilized egg.
In artificial embryo twinning, a researcher takes one of the embryo’s cells and replaces its nucleus with a piece of DNA from another cell, such as a skin cell. Then the cell is squirted into an empty egg, where it develops into a new embryo with identical DNA. The embryo can then be cut into pieces and studied.
Another way to make a clone is to use an electric current to smash the cells of an animal, such as a mouse or a dog. This is the method that scientists used to clone Dolly, the famous Scottish sheep.
But it’s hard to get the process exactly right. Sometimes extra DNA from the original cell is left behind. That could cause health problems in the future, especially if it is carried on through generations.
Scientists are working on ways to clone humans. But many people worry that clones would be unhealthy or unnatural. If someone created a clone of their favorite basketball player, that clone might feel pressure to be like the athlete, even if it doesn’t suit them. This is an ethical problem that needs to be thought about carefully before human cloning goes ahead.
5. Test the clone
Molecular cloning is a powerful tool that allows scientists to isolate specific DNA sequences. A typical cloning experiment inserts the target gene into a circular piece of DNA called a vector. The vector is then introduced into bacteria by a process called transformation. Bacteria carrying the plasmid are then selected using antibiotics. Those that are able to grow on a minimal medium containing lactose are likely to contain the target gene and can be identified by their blue color. A negative selection method, such as blue-white screening, can also be used to identify clones by examining their ability to synthesize lactose.
Although many people associate the term “cloning” with Dolly the sheep, cloning actually refers to making genetically exact copies. This can be done for genes, cells, and even whole organisms–but not humans (human cloning is currently illegal in most countries). Scientists have been cloning plants and animals for decades, with the cloning of human embryos occurring more recently.
Cloning involves placing a gene into a circular piece of DNA and then introducing it into bacteria, where the gene will be expressed and produce a protein. Alternatively, the DNA can be introduced into cells that have been emptied of their nucleus and then fertilized with another cell’s nucleus. Both techniques have a variety of applications in biological research.
This 6- to 8-wk project-based exercise, developed in partnership with Bio-Rad Laboratories, allows students to use a genomic DNA library of four different plant species and cultivars to isolate, clone, and characterize the GAPDH gene. The final product, a GenBank sequence for the GAPDH gene from a new species or variety not yet published in GenBank, will be submitted by students to the National Center for Biotechnology Information database.
