cdna 10k Archives - Gen9 Genetics
The first two chapters covered many important aspects of genes, such as how they function in heredity, how they code for proteins (broadly speaking), and their chemical nature. All of this was learned without having a single purified gene. A complete understanding of a gene, or the entire set of genes in a genome, requires that they be isolated and then intensively studied. Once a gene is "in hand", in principle, both its biochemical structures and its function (s) in an organism can be determined. One of the goals of biochemistry and molecular genetics is to assign particular functions to individual or compound structures. This chapter covers some of the techniques commonly used to isolate genes and illustrates some of the analyzes that can be performed on isolated genes.
Methods to purify some abundant proteins were developed in the early 1900s, and some of the experiments on the fine structure of the gene (gene and protein collinearity for trpA and tryptophan synthase) used microbial genetics and protein sequencing. However, methods for isolating genes were not developed until the 1960s and were applicable to only a few genes.
All of this changed in the late 1970s with the development of recombinant DNA or molecular cloning technology. This technique allowed the researchers to isolate any gene from any organism from which intact DNA (or RNA) could be isolated. The full potential to provide access to all genes in organisms is now being realized as entire genomes are sequenced. One of the by-products of the intense investigation of individual DNA molecules after the advent of recombinant DNA was a procedure to isolate any DNA whose sequence is known. This technique, called polymerase chain reaction (PCR), is much easier than traditional molecular cloning methods and has become a staple of many laboratories in the life sciences. After covering the basic techniques in recombinant DNA technology and PCR, its application to studies of the structure and function of eukaryotic genes will be discussed.
Like many advances in molecular genetics, recombinant DNA technology has its roots in bacterial genetics.
The first genes isolated were bacterial genes that could be taken up by bacteriophages. By isolating these hybrid bacteriophages, the DNA of the bacterial gene could be recovered in a highly enriched form. This is the basic principle behind recombinant DNA technology.
Some bacteriophages will integrate into a bacterial chromosome and reside in a latent state (Fig. 3.1). The DNA of the integrated phage is called a prophage and the bacterium is now a lysogen. The phages that do this are lysogenic. Induction of the lysogen will result in the cleavage of the prophage and multiplication to produce large progeny, that is, it enters a lytic phase in which the bacteria break open and are destroyed. The nomenclature is descriptive. Bacteria that carry the prophage do not show obvious signs of phage (except immunity to superinfection with the phage itself, which is explained later in part four), but when induced (for example, by stress or ultraviolet radiation) they will generate a lytic state, so they are called lysogens. Induced lysogens produce phage from the integrated prophage. Phages that always multiply when they infect a cell are called lytic.
Cleavage of a prophage from a lysogen is not always accurate. Typically, only phage DNA from the bacterial chromosome is cut, but occasionally some adjacent host DNA is included with the excised phage DNA and encapsulated in the progeny. These transducer phages are usually biologically inactive because part of the bacterial chromosome replaces part of the phage chromosome; these can be propagated in the presence of helper phages that provide the missing genes when co-infected with the same bacteria. When the transducer phage DNA is inserted into the newly infected cell, the bacterial genes can recombine on the host chromosome, thus bringing in new alleles or even new genes and genetically altering the infected cell. This process is called transduction.