Microbiologyprocedure.com Community Toolbar Download Image

Submit Your College, Institute, Company, Products for FREE

Index >>Bacterial Classification>>Genetic Methods in Taxonomy

Genetic Methods in Taxonomy

	The classical Adansonian approach of classifying microbes is based on phenotypic characteristics. Such characteristics are expressions of a large number of genes that control cellular activities through enzymes. It is now generally agreed that the phenotype is the reflection of the DNA base sequence. The best method of distinguishing two organisms should therefore be on the basis of composition of their genetic material. In recent years, the genetic characterization of organisms has been substantially developed. One is the analysis of the base composition of DNA i.e. to determine the mole per cent of guanine and cytosine in DNA (% G+C). The second, is to determine the degree of similarity between two DNA samples by hybridization between DNA and DNA or DNA and RNA.
The basis of this test is that the degree of hybridization would be Ian indication of the degree of relationship (homology). DNA of microorganisms contains four bases namely adenine (A), guanine (G), thymine (T) and cytosine (C) and in a double stranded DNA, A pairs with T and G pairs with C. he relative percentage of guanine and cytosine (G+C / A+T+G+C ) x 100
varies widely with different bacteria. The composition of chromosomal DNA is a fixed property of each cell and is independent of age and other external influences.The per cent (G+C) of chromosomal DNA can be determined by extracting DNA from cells by rupturing carefully. The DNA is then purified to remove non-chromosomal DNA. The base composition can then be determined either by subjecting the purified DNA to increasing temperature and determining theincrease in hypochromicity or by centrifugation of the DNA in cesium chloride density gradients. The basis of the first method i.e. the melting point method, is that when double stranded DNA is subjected to increasing temperature, the two DNA strands separate at a characteristic temperature. The melting temperature depends upon the G+C content of the DNA. Higher the G+C content, higher will be the melting point.
The mean temperature at which thermal denaturation of DNA occurs is called the melting point (Tm) and this is determined by noting the change in optical density of DNA solution at 260 nm during the heating period (Fig. 5.6). From the melting point, the mole per cent (G+C) can be calculated as below: Per cent G+C=Tm X 63.54/0.47
	The per cent (G+C) composition can also be calculated by determining the relative rate of sedimentation in a cesium chloride solution. DNA preparations when subjected to high gravitational force (as in a ultracentrifuge) in a heavy salt solution. will sediment at a region in the centrifuge tube where its density is equal to the density of the medium. By this method, DNA samples which are heterogenous can also be separated simultaneously. The buoyant density is very characteristic of each type of DNA and is dependent on the percent GC content, From the bouyant density one can ca1culate the percent GC content by using empherical formula

P= 1.660+0.00098 (% G.C)g.cm-3

A third method of determining per cent (G+C) is by the controlled hydrolysis of DNA with acids and separating and measuring the nucleotides by chromatography. This method is laborious but simple. The base composition of DNA from a variety of organisms determined by these procedures variety of organisms determined by these procedures.The genetic relatedness can also be determined by measuring the extent of hybridization between denatured DNA molecules between single stranded DNA and RNA species. The degree of homology is determined by mixing two kinds of single stranded DNA or single stranded DNA with RNA under appropriate conditions and then measuring the extent to which they associate to form double stranded structures. This can be precisely measured by making either the DNA or RNA radioactive .The degree of relatedness of different bacteria as determined by DNA-RNA hybridization. Although genetic relatedness can be determined by DNA-RNA hybridization, the DNA-DNA hybridization is most accurate provided precautions are taken to ensure that hybridization between two strands is uniform.

Home | Site map | Submit Article | Directory | Search

Language >> English | Français | Español | Deutsch | Italiano | Portugues | Japanese | Korean | Sim-Chinese