What does the chemical DNA look like, and how does one see it? DNA can be precipitated out of solution using alcohol and salt. The precipitate, which can be spooled out like long pasta, is clear if it is unbroken. DNA is colorless. Thus, when DNA molecules are separated by electrophoresis or hybridized with one another, we need to be able to see the location of the DNA. Two general methods are used to visualize or label DNA so that investigators can track it.
Fluorescently Staining DNA. Many dyes that fluoresce under UV light can be used to visualize or stain DNA; these are particularly useful during gel electrophoresis. The most commonly used stain is ethidium bromide (often given the abbreviation EtBr). During gel electrophoresis, EtBr is typically added to the gel before it solidifies. Ethidium bromide is a flat molecule with a ring structure that has a natural orange fluorescence under UV light. When added to a solution with double-stranded DNA, ethidium bromide stacks or intercalates itself between the base pairs. Upon its intercalation between the bases, the natural fluorescence of EtBr increases dramatically. This increased fluorescence is probably because the internal environment of DNA excludes water, and water quenches the fluorescence. Thus, when it intercalates in a reduced water environment, EtBr is unquenched and fluoresces more brightly. This bright fluorescence can be readily detected by UV and photographed.
Because the change in fluorescence requires stacking between the base pairs, EtBr is not widely used for visualizing single stranded molecules including RNA. RNA and single-stranded DNA can form intrastrand base pairs, so EtBr can be used for this but it is not very sensitive. EtBr staining is probably the one molecular biology tool that depends on the most familiar property of DNA, namely the double helix.
While EtBr is widely used and very inexpensive, the potential hazards associated with its use have led to the development of other dyes. These include SYBR dyes that are more sensitive than EtBr and so can detect lower concentrations of DNA. Their toxicity is reported by their manufacturers to be less hazardous than EtBr. The SYBR dyes are considerably more expensive than EtBr so most labs use EtBr for routine staining of DNA during gel electrophoresis.
Labeling DNA by sequence. EtBr and related molecules stain DNA non-specifically so any double-stranded nucleic acid can be seen. This is useful for detecting the presence of DNA, such as for molecules that have been resolved by size on a gel, but it does not detect specific sequences. Thus, methods have been developed to make it is possible to label DNA molecules of specific sequences, and a variety of different labels and methods for detection have been developed. Nearly all of these methods begin with a process for synthesizing or replicating DNA in vitro. DNA replication requires the enzyme DNA polymerase, a short nucleotide or deoxynucleotide sequence to act as a primer for the reaction, appropriate salts, and the four deoxynucleotides (the dNTPs). Since the specificity of DNA functions depend on its base sequence, methods for labeling specific sequences depend on labeling these dNTPs.
In an in vitro labeling reaction, one of the four dNTPs has a label attached to it. For example, in older experiments, the dTTP solution used for the reaction included some dTTP molecules radioactively labeled with 32P at the α-phosphate position. dTTP was used to prevent the accidentally labeling of RNA molecules; RNA has uracil rather than thymidine. Thus, whenever the copy of the DNA molecule was synthesized in vitro, radioactively labeled thymidine could be inserted rather than regular thymidine. Not every dTTP nucleotide had the radioactive label, so some thymidines are not labeled. As a result, every newly synthesized DNA molecule made in vitro could be radioactively labeled.
Radioactive labels have been largely replaced by a variety of non-radioactive labels that are safer to use but the same basic procedure is used. One of the dNTPs is labeled with some molecule that does not affect its interactions and functions, so that the synthesized DNA can be recognized. Among the other labels that are in widespread use are the fluorescent dyes Cy3 and Cy5, and the small molecule biotin. Cy3 fluoresces in the yellow-green range, while Cy5 fluoresces in the red range. Biotin (also known as vitamin B7) is a small molecule that is “avidly” and specifically bound by the bacterial protein streptavidin. Streptavidin can be purchased with many different tag molecules on it, so the interaction of the labeled streptavidin with the biotinylated DNA molecule indicates the presence of the DNA.