Essential Tools for Protein Engineering
Gene Cloning and Other Molecular Genetics Techniques
Gene cloning is the act of making copies, or clones, of a single gene. Once a gene is identified, clones can be used in many areas of biomedical and industrial research. Genetic engineering is the process of cloning genes into new organisms or altering the DNA sequence to change the protein product. Genetic engineering depends on our ability to perform certain essential procedures.
A common use for gene cloning is the development of genetically modified crops in agriculture. Foreign genes with the desired trait might be inserted into a particular crop to achieve the desired result.
Gene cloning is also an important part of stem cell research. Stem cells can turn into any type of cell, meaning that researchers can use them to grow necessary tissues or organs.
In vitro gene cloning uses the polymerase chain reaction (PCR) method to copy DNA fragments. In vivo gene cloning copies fragments into host cells through the use of restriction enzymes and ligases using vectors.
The discovery of thermostable DNA polymerases, such as Taq Polymerase, made it possible to manipulate DNA replication in the laboratory and was essential to the development of PCR. Primers specific to a particular region of DNA, on either side of the gene of interest, are used, and replication is stopped and started repetitively, generating millions of copies of that gene. These copies can then be separated and purified using gel electrophoresis.
The discovery of enzymes known as restriction endonucleases has been essential to protein engineering. These enzymes cut DNA at specific locations based on the nucleotide sequence. Hundreds of different restriction enzymes, capable of cutting DNA at a distinct site, have been isolated from many different strains of bacteria. DNA cut with a restriction enzyme produces many smaller fragments of varying sizes. These can be separated using gel electrophoresis or chromatography.
Purifying DNA from a cell culture or cutting it using restriction enzymes wouldn't be of much use if we couldn't visualize the DNA—that is, find a way to view whether your extract contains anything, or what size fragments you've cut it into. One way to do this is by gel electrophoresis. Gels are used for a variety of purposes, from viewing cut DNA to detecting DNA inserts and knockouts.
Join Two Pieces of DNA
In genetic research, it often is necessary to link two or more individual strands of DNA to create a recombinant strand or close a circular strand that has been cut with restriction enzymes. Enzymes called DNA ligases can create covalent bonds between nucleotide chains. The enzymes DNA polymerase I and polynucleotide kinase are also important in this process for filling in gaps or phosphorylating the 5-foot ends, respectively.
Selection of Small Self-Replicating DNA
Small circular pieces of DNA that are not part of a bacterial genome but are capable of self-replication are known as plasmids. Plasmids are often used as vectors to transport genes between microorganisms. In biotechnology, once the gene of interest has been amplified and both the gene and plasmid are cut by restriction enzymes, they are ligated together generating what is known as a recombinant DNA. Viral (bacteriophage) DNA can also be used as a vector, as can cosmids, which are recombinant plasmids containing bacteriophage genes.
Method to Move a Vector Into a Host Cell
The process of transferring genetic material on a vector such as a plasmid, into new host cells, is called transformation. This technique requires that the host cells are exposed to an environmental change that makes them "competent" or temporarily permeable to the vector. Electroporation is one such technique. The larger the plasmid, the lower the efficiency with which it is taken up by cells. Larger DNA segments are more easily cloned using bacteriophage, retrovirus, or other viral vectors or cosmids in a method called transduction. Phage or viral vectors are often used in regenerative medicine but may cause insertion of DNA in parts of our chromosomes where we don't want it, causing complications and even cancer.
Methods to Select Transgenic Organisms
Not all cells will take up DNA during transformation, but scientists need a method for detecting the ones that do. Generally, plasmids carry genes for antibiotic resistance, and transgenic cells can be selected based on expression of those genes and their ability to grow on media containing that antibiotic. Alternative methods of selection depend on the presence of other reporter proteins such as the x-gal/ lacZ system, or green fluorescence protein, which allow selection based on color and fluorescence, respectively.