In any method of extraction and purification, there are three main steps:
1. Breaking of the cells
2. Extraction of DNA
3. Purification
Cells can be broken in different ways. One common method for lysis of bacterial cultures is alkaline lysis. In the case of animals, cells can be lysed by simple detergents or by hypotonic solutions. Plant tissues can be homogenized by strong detergents such as SDS and heating at high temperatures. But there are various types of DNA isolation kits marketed by a number of biotechnology companies, which are very simple, short, and easy to handle.
Isolation of Plasmid DNA by Alkaline Lysis Method:
This method is used for the large-scale isolation of plasmid and cosmid DNA by a modification of alkaline lysis procedure, followed by purification by phenol chloroform extraction. Cells containing the desired plasmid or cosmid are harvested by centrifugation, incubated in a lysozyme buffer (re-suspension buffer), and treated with alkaline detergent. The alkali breaks the cells and the DNA and proteins- are released into the medium. Detergent solubilizes the proteins and DNA. The proteins and membranes are precipitated with sodium acetate. The precipitate is centrifuged out at a higher RPM and the supernatant contains the DNA. Finally, the DNA is precipitated out by adding 95%. ethyl alcohol or propanol. The DNA pellet is resuspended in a Tris EDTA buffer. This DNA sample contains some DNA-binding proteins, which have to be removed. This can be carried out by phenol-chloroform extraction. There are several variations to this protocol, which is suited to the situations- and type of bacterial cultures.
Genomic DNA Isolation from Blood:
Genomic DNA isolation is performed according to the standard protocol suggested by Federal Bureau of Investigation, USA. After the blood samples (stored at -70°C in EDTA vacutainer tubes) are thawed, a standard citrate buffer is added, mixed, and the tubes are centrifuged. The top portion of the supernatant is discarded and additional buffer is added, mixed, and again the tube is centrifuged. After the supernatant is discarded, the pellet is resuspended in a solution of SDS detergent and proteinase K, and the mixture is incubated at 55°C for one hour. The sample then is phenol-extracted once with a phenol/ chloroform/ isoamyl alcohol solution, and after centrifugation the aqueous layer is removed to a fresh microcentrifuge tube. The DNA is ethanol-precipitated, resuspended in buffer, and then ethanol-precipitated a second time. After the pellet is dried, buffer is added and the DNA is resuspended by incubation at 55°C overnight, and the genomic DNA solution is assayed by the polymerase chain reaction.
DNA Isolation from Plant Tissues:
Plant tissues bring up several problems during DNA isolation. Plant cells have a rigid cell wall and the tissue contains a number of toxic metabolites, which can interact with the DNA and change its nature and make it useless for other experimental purposes. Metabolites such as mucilage and other carbohydrates can very easily- form complexes with DNA and it can be damaged. Therefore, the extraction buffer should be supplemented with some compounds that can protect the DNA against these metabolites.
Many DNA-isolation techniques widely employed by plant molecular biologists use a CTAB (Cetyltrimethylammonium bromide) extraction buffer. This compound forms a complex with DNA and thus protects it from other toxic metabolites such as mucilage and phenolics.
The DNA, isolated and purified by any of these methods, can be used for a variety of experimental purposes. It can be used for restriction digestion analysis, cloning, ligation, transformation experiments, in vitro transcription, PCR amplification, RFLP (restriction length polymorphism), fingerprinting, RAPID (random amplification polymorphic DNA), sequencing, nick translation and radiolabeling, preparation of genomic DNA library and cDNA library, etc.
Tags: Bio Technology, Bio Genetics, DNA Isolation
0 comments:
Post a Comment