Microbiology Procedure

Index >> Microbial Genetics >> Isolation of DNA by Shotgun Method

Isolation of DNA by Shotgun Method

	Isolation of DNA by Shotgun Method - The DNA of an organism may contain from a few to thousands of genes. The problem, therefore, is , to isolate the required gene from the entire DNA of the organism. If the property of a particular gene is easily detected, the 'shotgun' technique can be used to isolate the gene. The other approach is to isolate the messenger RNA (mRNA) transcribed by the gene and then obtain DNA from it. I. The 'shotgun' method: In this method the total DNA of the organism is subjected tothe action of restriction enzymes and broken down into many fragments. Each of these fragments is then combined with the DNA from a suitable vector to produce recombinant DNA.
The different recombinants are inserted at random into a host cell, usually E.coli The different types of recombinant host cells are cultured separately and grown into colonies. The details of this method will now be taken up 1. DNA is isolated from the organism of choice (virus, bacterium , plant or animal) and is cleaved by restriction enzymes such as EcoRI into many fragments. Such DNA is called 'foreign' DNA, and is designated as X DNA . The DNA, restriction enzyme cuts the DNA at specific points, leaving overlapping breaks with 'sticky' (cohesive) ends. The fragments (X 1 to X 4) are of different lengths.
2. The outer membrane of E. coli bacteria (host cells) is first dissolved by a detergent like liquid and its DNA is released. The plasmids are separated from chromosomal DNA in an ultracentrifuge. They are now placed in a solution containing the same restriction enzyme which cuts the plasmid ring (pDNA) into a linear form. Linear plasmid DNA also has overlapping., single-stranded, cohesive ends. 3. Foreign DNA fragment's are mixed with linear plasmid DNA. Random association between the two types of DNA takes place by hydrogen bonding at the cohesive ends. The foreign DNA becomes inserted into plasmid DNA to form a larger ring or a longer linear form.
Covalent joining is brought about by another enzyme, DNA ligase. The resulting DNA is called a plasmid chimera or recombinant DNA. Both linear and circular molecules of plasmid DNA are joined to differt1nt combinations of fragments. 4. The chimera plasmids are placed in a solution containing cold calcium chloride and normal E.coli bacteria. On heating suddenly (2-5, minutes, 42°C) the E.coli membranes become permeable to plasmid chimeras, which pass into the cell. Thus recombinant DNA plasmids are taken up by E.coli cells.
	5. The bacterial cells are spread on a nutrient medium in a Petri dish. Each E.coli cell, containing particular genes inserted in its plasmids, grows by repeated binary fission into a colony. The individuals of each colony are all derived from one E.coli cell, and are hence genetically identical. Such groups of genetically identical individuals are caned clones Each colony contains 'individuals with specific segments of foreign DNA in their plasmids. Even under favorable conditions only one cell in 105 or 106 produces a transformed clone. By using the shotgun method the genomes of several organisms (rRNA gene of the toad Xenopus laevis, the histone gene of sea-urchin, 90% of the genome of E.coli, yeast, Drosophila) have been cloned into E.Coli

Home | Site map | Submit Article | Resources | Search

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