Tools and Settings
Content
Questions and Tasks
Gene cloning involves removing a gene from the genome of an organism and then placing that isolated gene into the genome of a bacterial cell. The bacterial cell is then responsible for maintaining this foreign gene. For examples, the bacterial cell copies the foreign gene by DNA replication, the bacterial transcribes the foreign gene to make a messenger RNA (mRNA) molecule, and the bacterial cell translates the mRNA to make a protein product. Many important human proteins, including insulin (for diabetes patients) and factor VIII (for type A hemophilia patients), have been produced in large quantities by bacterial cells via this gene cloning technique.
In the early 1970s, scientists isolated the DNA from two different organisms and covalently linked them together to form a hybrid DNA molecule in a test tube. This new hybrid DNA molecule, which contained DNA from two sources, is a recombinant DNA molecule. This experiment demonstrated the first use of recombinant DNA techniques, methods to manipulate DNA molecules outside of a living organism. Since then, many advances have made recombinant DNA techniques common practice among biologists.
Our discussion will focus on a process called gene cloning (figure 12.1). Gene cloning involves isolating a particular gene of interest and inserting that gene into a vector DNA molecule. Commonly used vectors include the circular plasmid DNA molecules found in many bacteria. The resulting recombinant DNA molecule, composed partly of the gene of interest and partly the plasmid vector, is then introduced into a host bacterial cell by transformation. The host bacterial cell maintains the recombinant DNA molecule, so the gene of interest (i.e., the cloned gene) can be studied in more detail. Once gene cloning is complete, the cloned gene can be used in:
As mentioned earlier, gene cloning involves removing a gene of interest from an organism and inserting that gene into a vector DNA molecule (figure 12.2). The resulting recombinant DNA molecule is then introduced into a host organism for maintenance. The host organism is often the bacterium Escherichia coli.
From this point on, let us assume that we are interested in studying the insulin gene isolated from the human genome. The overall goal of our gene cloning experiment is to insert the human insulin gene into a vector DNA molecule and introduce the recombinant DNA molecule into the bacterium E. coli. In our scenario, the vector DNA molecule will function to:
Plasmids, small circular DNA molecules found in many bacteria, often serve as vector molecules (figures 12.1 and 12.2). These plasmids are not part of the bacterial chromosome. Plasmids contain:
How do we take the insulin gene from a human chromosome and insert it into a plasmid vector DNA molecule?
Restriction enzymes are important molecule tools used in the gene cloning process. Restriction enzymes are endonucleases that recognize specific DNA sequences (restriction enzyme sites) and cleave the phosphodiester bonds within both DNA strands. The restriction enzyme site is typically a palindrome DNA sequence. For example, the restriction enzyme EcoRI isolated from the bacterium E. coli cuts the DNA sequence 5’-GAATTC-3’. The complementary strand is 3’-CTTAAG-5’, which is identical to the original restriction enzyme sequence but in the reverse orientation (i.e., a palindrome). EcoRI cuts both DNA strands within this palindromic DNA sequence between the G and A nitrogenous bases.
The natural function of a restriction enzyme is to protect the bacterial cell from foreign DNA, particularly bacteriophage DNA injected into the bacterial cell during a phage infection. Several hundred restriction enzymes have been isolated from bacteria and are available commercially for purchase.
How can we use the restriction enzyme EcoRI to clone the human insulin gene into a plasmid vector DNA molecule?
Suppose that the restriction enzyme EcoRI recognizes the restriction enzyme sites shown below in both the insulin gene and in the plasmid DNA molecule (see figure 12.3) Note that the plasmid is cut by EcoRI at a single site, while EcoRI cuts on both ends of the human insulin gene (the target gene in the figure 12.3).
EcoRI cleaves the cloning site within the plasmid and the insulin gene to produce complementary single-stranded regions called sticky ends. When mixed, the sticky ends of the insulin DNA form hydrogen bonds with the sticky ends from the vector DNA. DNA ligase then forms the final covalent bonds in each DNA strand, covalently linking the insulin gene into the plasmid vector.
The entire procedure used to clone the insulin gene into a plasmid vector is outlined below (figure 12.4). For the purposes of this hypothetical experiment, let us assume that the plasmid DNA molecule contains a restriction enzyme site (cloning site), the ampR gene as a selectable marker, OriC, and the lacZ gene. The function of the lacZ gene will be described below.
How do we determine if β-galactosidase is produced? This is done by plating the bacterial cells on an agar plate that not only includes ampicillin but also IPTG, a chemical that activates the lacZ gene to produce the β-galactosidase protein. The agar plate also contains X-Gal, a chemical substrate for the β-galactosidase enzyme.