Restriction-Modification Systems of Bacteria
Southern and Northern Analysis
Polymerase Chain Reaction (or PCR)
Southern and Northern Hybridiazation Analysis
The hybridization or binding of a clone to DNA or RNA provides important information regarding the structure and the expression of the gene. Southern hybridizations involve the binding of a radioactive probe to a DNA molecule that is immobilized on a membrane filter. After a series of washes the filter is used to expose a piece of X-ray film. After exposure, the film is developed, and a band appears where the hybridization occurs. Each of these hybridizations will represent the size of fragments which contain sequences that are complementary to the probe. By hybridizing the probe to a series of restriction digestions of the DNA of interest, the structure of the gene can be analyzed.Let's assume that your gene does not contain any internal EcoRI sites. Then if you hybridize a clone (for example a cDNA clone for some gene) to an EcoRI digestion of your DNA and you see two fragments, then you can conclude that the species you are analyzing has two copies of the gene. Normally, you do not know a priori what restriction enzyme sites are located in the gene, so a series of digestions and hybridizations are performed. If five out the six experiments reveal two bands, and the probe hybridizes to three fragments of DNA digested with a sixth, then you can conclude that two genes exist and one of these genes contains a restriction cleavage site for the third enzyme.
Northern hybridizations involve a radioactive probe and RNA that is immobilized on a filter membrane. The hybridization is between complementary bases in the RNA and the probe. These hybridizations are performed to study the expression of the gene. RNA is typically isolated from different tissues and from different developmental stages of species. After electrophoresis, the RNA is transferred to the membrane and probed. If, for example, the probe hybridizes only to RNA from heart tissue after the individual reaches adult age, it can be concluded that the gene is only expressed in the adult heart.
In general, genes can be expressed constitutively, temporally or spatially. Constitutive expression implies that the gene is expressed at all times. mRNA for genes that exhibit spatial expression are only found in specific tissues of the organism. If a gene is only expressed during a specific time in development it is said to exhibit temporal expression. Combinations of expression patterns are also possible. For example, a gene that is always expressed in a leaf tissue is exhibiting constitutive, spatial expression.
Copyright © 1997. Phillip McClean