Assessment Of Quality And Quantity

In many cases, purified nucleic acid is used directly in an assay, without evaluation of its purity, concentration, or size. The results of the assay itself often demonstrate the success or failure of the preparation. In cases where assay setup or interpretation requires prior knowledge of the sample's purity and concentration, a number of methods are available.

Perhaps the simplest and fastest approach is spectrophotom-etry. Although nucleic acid bases show maximal absorption at an approximate wavelength of 260 nm, contaminating proteins absorb well at 280 nm. Protein absorption is primarily the result of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Testing sample optical density (OD) at 260 nm and 280 nm, therefore, allows assessment of both nucleic acid concentration and purity. An OD260 of 1.0 corresponds to approx 50 |g/mL of double-stranded DNA (40 |g/mL for single-stranded DNA or RNA). The OD260/280 ratio provides an estimate of nucleic acid purity, with a pure preparation having a ratio between 1.8 and 2.0. Contamination with organic solvents or protein will obviously lower this value and also prevent accurate nucleic acid quantification from the OD260 reading.

If the purified sample is too contaminated or low in concentration for spectrophotometric assessment, fluorescent dyes can be used to quantify the nucleic acid present. A variety of dyes that bind nucleic acid are available, including acridine orange, daminoibenzoic acid (DABA), propidium iodide, and ethidium bromide. These dyes might detect nucleic acid in quantities as low as 1-5 ng (24), although, typically, the detection threshold is closer to 5-10 ng (29). Ethidium bromide, most widely used of these compounds, demonstrates fluorescence that increases by a factor of 20 or more when bound to nucleic acid. This fluorescence under ultraviolet (UV) light is directly proportional to the amount of nucleic acid present. The direct relationship between fluorescence and nucleic acid content allows the use of ethidium bromide in quantitative assays. It is worth noting that the degree of fluorescence is dependent on the ratio of ethidium bromide to nucleic acid, with maximal output at a DNA : ethidium bromide ratio of 0.5 to 3.0 (w/w) (30).

Nucleic acid samples and standards (of known concentration) can be spotted with ethidium bromide on a plastic surface, and fluorescence can be captured by digital or conventional camera. Some contaminants might increase or decrease fluorescence, but this can be partly remedied by spotting on a 1% agarose slab. Small, interfering molecules will diffuse away from nucleic acid over the course of a short incubation at room temperature (31).

Further information can be obtained by electrophoretic analysis of the purification product. As discussed earlier, the size of nucleic acid fragments can be important to subsequent work. Small agarose gels, "minigels," can be used easily and quickly to determine both the size and quantity of nucleic acids. For size determination, a molecular-weight ladder provides a reference standard. Likewise, for quantifying nucleic acid, a series of samples of known concentration can be run in parallel with the unknown sample, and a standard curve prepared. Again, a fluorescent dye like ethidium bromide should be used to detect nucleic acid in the gel, so that nucleic acid quantity can be extrapolated from fluorescence data.

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