Nucleic Acid Measurement for Quantity and Quality

Nucleic acid quantitation is optional for many protocols that utilize in vitro nucleic acid amplification. Some methods, however, require use of more accurate quantities of nucleic acid, so assessment of the yield and concentration of purified nucleic acids is useful. This is typically done using ultraviolet (UV) spectrophotometry. The absorbance of a nucleic acid solution is measured at several wavelengths. The maximal absorbance for nucleotides is at 260nm of UV light (A260), while for proteins the maximal absorbance is at 280 nm (A280). Nucleic acids can therefore be quantified by the A260 measurement, while the A260/A280 ratio provides an estimate of the purity of the sample. Pure DNA has an A260 of 1.0 at a concentration of 50 |ig/ml and an A260/A280 ratio of 1.8, while pure RNA has an A260 of 1.0 at a concentration of 40 |g/ml and an A260/A280 ratio of 2.0. Lower A260/A280 ratios indicate the presence of protein in the solution. Other contaminants can be detected by their absorbance at other wavelengths, such as phenol at A270 and guanidinium at A230.

Ethidium bromide (EtBr) intercalates into DNA strands, causing DNA to fluoresce upon illumination with UV light. The fluorescence of EtBr correlates with the number of base pairs of DNA in which the EtBr is intercalated, which is a result of both the size and quantity of the DNA fragment. Therefore, by staining sample DNA with EtBr in an electrophoresis gel and comparing the brightness to mass standards in adjacent lanes, the quantity of DNA can be estimated. This provides a convenient system for quantification of post-PCR DNA prior to sequencing, since the UV spectrophotometer is usually kept in the pre-PCR area (see below for PCR, sequencing, and amplicon carryover contamination). More important, the image of the EtBr-stained sample DNA can be used to assess DNA quality. High-quality, substantially intact DNA forms a single band close to the well serving as the origin of elec-trophoresis. In contrast, DNA degradation is apparent as a smear of EtBr-stained DNA extending downward from the well. Ethidium bromide is mutagenic and produces light background staining and is therefore being replaced by other intercalating dyes such as SYBR Green.

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