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Chapter: Biotechnology Applying the Genetic Revolution: Recombinant DNA Technology

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DNA Isolation and Purification

Basic to all biotechnology research is the ability to manipulate DNA. First and foremost for recombinant DNA work, researchers need a method to isolate DNA from different organisms.

DNA ISOLATION AND PURIFICATION

Basic to all biotechnology research is the ability to manipulate DNA. First and foremost for recombinant DNA work, researchers need a method to isolate DNA from different organisms. Isolating DNA from bacteria is the easiest procedure because bacterial cells have little structure beyond the cell wall and cell membrane. Bacteria such as E. coli are the preferred organisms for manipulating any type of gene because of the ease at which DNA can be isolated. E. coli maintain both genomic and plasmid DNA within the cell. Genomic DNA is much larger than plasmid DNA, allowing the two different forms to be separated by size.

To release the DNA from a cell, the cell membrane must be destroyed. For bacteria, an enzyme called lysozyme digests the peptidoglycan, which is the main component of the cell wall. Next, a detergent bursts the cell membranes by disrupting the lipid bilayer. For other organisms, bursting the cells depends on their architecture. Tissue samples from animals and plants have to be ground up to release the intracellular components. Plant cells are mechanically sheared in a blender to break up the tough cell walls, and then the wall tissue is digested with enzymes that break the long polymers into monomers. DNA from the tail tip of a mouse is isolated after enzymes degrade the connective tissue. Every organism or tissue needs slight variations in the procedure for releasing intracellular components.

Once released, the intracellular components are separated from the remains of the outer structures by either centrifugation or chemical extraction. Centrifugation separates components according to size, because heavier or larger molecules sediment at a faster rate than smaller molecules. For example, after the cell wall has been digested, its fragments are smaller than the large DNA molecules. Centrifugation causes the DNA to form a pellet, but the soluble cell wall fragments stay in solution. Chemical extraction uses the properties of phenol to remove unwanted proteins from the DNA. Phenol is an acid that dissolves 60% to 70% of all living matter, especially proteins. Phenol is poorly water soluble, and when it is mixed with an aqueous sample of DNA and protein, the two phases separate, much like oil and water. The protein dissolves in the phenol layer and the nucleic acids in the aqueous layer. The two phases are separated by centrifugation, and the aqueous DNA layer is removed from the phenol.

Once the proteins are removed, the sample still contains RNA along with the DNA. Because this is also a nucleic acid, it is not soluble in phenol. Luckily, the enzyme ribonuclease (RNase) digests RNA into ribonucleotides. Ribonuclease treatment leaves a sample of DNA in a solution containing short pieces of RNA and ribonucleotides. When an equal volume of alcohol is added, the extremely large DNA falls out of the aqueous phase and is isolated by centrifugation. The smaller ribonucleotides stay soluble. The DNA is ready for use in various experiments.


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