DNA Extraction Research Paper

DNA extraction is a technique for isolating DNA from cells or tissues. Thus, extracted DNA can then be used in molecular biology research, such as sequencing, PCR, and cloning.

There are different protocols for extracting DNA, following roughly the same schematic:

  • Cell lysis
  • Removal of proteins
  • Elimination of other nucleic acids (RNA, etc.)
  • Concentration of DNA by alcohol precipitation

Different variants are used, depending on whether it is sought to extract genomic DNA (from the analyzed cells chromosomes) or plasmid DNA (from plasmids worn most often by bacterial cells such as Escherichia coli). There are now commercial kits for quickly achieving these extractions using ready to use reagents.

It starts in general by a lysis of cells or tissues, followed by extraction using detergents, which will disperse the lipid bilayers of membranes and denature proteins, particularly those associated with DNA in chromatin. The solution obtained is generally very viscous, because thus released DNA forms very long filaments opposed to hydrodynamic flow.

The next step is the deproteinization of the solution, which is done by extraction using organic solvents, generally of phenol with addition of some chloroform. The denatured proteins form a precipitate in the phenol-water interface, while DNA remains in solution in the aqueous phase that is recovered by sedimentation or centrifugation.

The DNA is then precipitated by adding ethanol or isopropanol in the aqueous phase, collected by centrifugation and dissolved in buffer. To eliminate traces of phenol and other contaminants, you can finally do dialysis or chromatographic purification step.

The plasmid DNA preparation from bacteria is one of the most common techniques in molecular biology, also known by such abbreviated names as miniprep, midiprep, and maxiprep depending on the volume of bacterial culture used. This principle of extraction is known as a rapid alkaline extraction procedure for screening recombinant plasmid DNA. This method allows to selectively prepare the plasmid DNA in bacteria, while removing the DNA from the bacterial chromosome.The principle of this method is to perform lysis of the cells using a detergent (sodium dodecyl sulfate) in the presence of soda to pH13. At this very alkaline pH, DNA is denatured, meaning that the two strands of the double helix are separated. It neutralizes then quickly the solution, which causes sudden renaturation (renaturation of the strands of the DNA duplexes). Very long DNA chromosome is unable to be re-matched completely and form insoluble tangles, while sahort plasmid DNA manages to reform and remains in solution, is then separated by centrifugation. Precipitated protein is also removed with detergent and chromosomal DNA. Plasmid DNA is then concentrated by precipitation with alcohol.

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