Objectives

• Compare and contrast organic, inorganic, and solid-phase approaches for isolating cellular and mitochondrial DNA.

• Note the chemical conditions in which DNA precipitates and goes into solution.

• Compare and contrast organic and solid-phase approaches for isolating total RNA.

• Distinguish between the isolation of total RNA with that of messenger RNA.

• Describe the gel-based, spectrophotometry, and fluoro-metric methods used to determine the quantity and quality of DNA and RNA preparations.

• Calculate the concentration and yield of DNA and RNA from a given nucleic acid preparation.

The purpose of extraction is to release the nucleic acid from the cell for use in subsequent procedures. Ideally, the target nucleic acid should be free of contamination with protein, carbohydrate, lipids, or other nucleic acid, i.e., DNA free of RNA or RNA free of DNA. The initial release of the cellular material is achieved by breaking the cell and nuclear membranes (cell lysis). Lysis must take place in conditions that will not damage the nucleic acid. Following lysis, the target material is purified, and then the concentration and purity of the sample can be determined.

Isolation of DNA

Although Miescher first isolated DNA from human cells in 1869,1 the initial routine laboratory procedures for DNA isolation were developed from density gradient centrifugation strategies. Meselson and Stahl used such a method in 1958 to demonstrate semiconservative replication of DNA.2 Later procedures made use of the differences in solubility of large chromosomal DNA, plasmids, and proteins in alkaline buffers. Large (>50 kbp) chromosomal DNA and proteins cannot renature properly when neutralized in acetate at low pH after alkaline treatment, forming large aggregates instead. As a result, they precipitate out of solution. The relatively small plasmids return to their supercoiled state and stay in solution. Alkaline lysis procedures were used extensively for extraction of 1-50-kb plasmid DNA from bacteria during the early days of recombinant DNA technology.

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