METHODS OF DETECTION FOR NUCLEIC ACIDS
Recombinant DNA methodologies require the ability to detect DNA. One of the easiest ways to detect the amount of DNA or RNA in solution is to measure the absorbance of ultraviolet light at 260 nm (Fig. 3.4). DNA absorbs ultraviolet light because of the ring structures in the bases. Single-stranded RNA and free nucleotides also absorb ultraviolet light. In fact, they absorb more light because their structures are looser. Since the absorbance of UV light depends on the amount of DNA and the molecular structure, the relationship between UV absorbance and concentration is Double-stranded DNA concentration (μg/ml) = (OD260) × (50 μg DNA/ml)/ (1 OD260 unit) RNA concentration (μg/ml) = (OD260) × (40 μg RNA/ml)/(1 OD260 unit) In addition to the amount of DNA, a second absorbance reading at 280 nm is commonly used to determine the purity of the sample. The ratio of the 260 nm absorbance value divided by the 280 nm absorbance value will indicate whether the sample is pure. If the sample is pure DNA, then the 260/280 ratio is 1.8; whereas a 260/280 ratio for pure RNA is 2.0. When the ratios deviate from the expected value, there could be residual phenol from the purification or a very low concentration of DNA or RNA.
The concentration of DNA or RNA in a liquid can be determined by measuring the absorbance of UV light at 260 nm.
FIGURE 3.4 Determining the Concentration of DNA All nucleic acids absorb UV light via the aromatic rings of the bases. Stacked nucleotides (on the left) absorb less UV than scattered bases (on the right) because of the ordered structure.
Radioactive Labeling of Nucleic Acids and Autoradiography
Ultraviolet light absorption is a general method for detecting DNA but does not distinguish between different DNA molecules. DNA can also be detected with radioactive isotopes (Fig. 3.5). During replication, radioactive precursors such as 32P in the form of a phosphate group and 35S in the form of phosphorothioate can be incorporated. Because native DNA does not contain sulfur atoms, one of the oxygen atoms of a phosphate group is replaced with sulfur to make phosphorothioate. Most radioactive molecules used in laboratories are short lived. 32P has a half-life of 14 days and 35S has a half-life of 68 days, so the isotopes degrade fairly fast. Although radioactive DNA is invisible, photographic film will turn black when exposed to the radioactive DNA. Radioactively labeled DNA is considered “hot,” whereas unlabeled DNA is considered “cold.” Autoradiography identifies the location of radioactively labeled DNA in the gel (Fig. 3.6). If the gel is thin, like most polyacrylamide gels, it is dried with heat and vacuum. If the gel is thick, like agarose gels, the DNA is transferred to a nylon membrane using capillary action (see Fig. 3.9, later). The dried gel or nylon membrane is placed next to photographic film. As the radioactive phosphate decays, the radiation turns the photographic film black. Only the areas next to radioactive DNA will have black spots or bands. The use of film detects where the hot DNA is on a gel, and the use of ethidium bromide shows where all of the DNA, hot or cold, is. These two methods allow distinguishing one DNA fragment from another.