Fluorescence in the Detection of DNA and RNA

Most classic work in biochemistry and molecular biology was done with radioactive tracers and probes. Although the low levels of radioactivity used in laboratory analyses are little real hazard, the burden of storage and proper disposal of the waste has made it relatively cheaper and quicker to use other detection methods. Some of the newer DNA detection methods use fluorescence, chemical tagging and hybridization.

Fluorescence occurs when a molecule absorbs light of one wavelength and emits light of lower energy at a longer wavelength (Fig. 21.22). Detection of fluorescence requires both a beam of light to excite the dye and a photo-detector to detect the fluorescent emission. Fluorescent dyes can be attached to DNA molecules and modern automated methods for DNA sequencing make use of such fluorescent tagging (see

Fluorescence Process in which a molecule absorbs light of one wavelength and then emits light of another, longer, lower energy wavelength

A. Fluorescent tagging of dna

FIGURE 21.22 Fluorescence Detection

(A) Fluorescent tagging of DNA. During DNA synthesis, a nucleotide linked to a fluorescent tag is incorporated at the 3' end of the DNA. A beam of light excites the fluorescent tag, which in turn releases light of a longer wavelength (fluorescence).

(B) Energy levels in fluorescence. The fluorescent molecule attached to the DNA has three different energy levels, S0, S1', and S1. The S0 or ground state is the state before exposure to light. When the fluorescent molecule is exposed to a light photon of sufficiently short wavelength, the fluorescent tag absorbs the energy and enters the first excited state, S1'. Between S1' and S1, the fluorescent tag relaxes slightly, but doesn't emit any light. Eventually the high-energy state releases its excess energy by emitting a longer wavelength photon. This release of fluorescence returns the molecule back to the ground state.

B. Energy levels in fluorescence

Ch. 24). In this case, each of the nucleotide bases is labeled with a different fluorescent dye. As each of the bases pass through the sequencing machine, a laser activates the dye which fluoresces by emitting light of a lower wavelength. A detector records the wavelength of emitted light, and translates the data to give the identity of each passing base. The nucleotide sequence is printed for the researcher.

Another instrument that uses fluorescence is the Fluorescence Activated Cell Sorter or FACS. Its job, originally, was sorting cells labeled with a fluorescent tag from those that were untagged. The new generation of more sensitive FACS machines is capable of sorting chromosomes labeled by hybridization with fluorescent tagged DNA probes (Fig. 21.23). Another novel use for a FACS machine is in sorting whole small organisms such as the nematode worm C. elegans, which is widely used in genetic analysis. Gene expression may be monitored by making gene fusions to green fluorescent protein (see Ch. 25 for gene fusion analysis). Consequently, organisms with different levels of fluorescence are produced and may need to be screened by automatic sorting if generated in large numbers.

Another recent and growing use for FACS technology is in fluorescent bead sorting. Many reactions in modern high-throughput screening involve anchoring one or more reagents to microscopic polystyrene beads. In some reaction schemes, fluo-rescently labeled molecules may bind to colorless reagents, such as DNA or proteins, previously attached to the beads. In other cases, the beads may be color coded by fluorescent dyes before reaction occurs. In either case, the beads are sorted after fluorescence activated cell sorter (FACS) Instrument that sorts cells (or chromosomes) based on fluorescent labeling

Chemical Tagging with Biotin or Digoxigenin 587

FIGURE 21.23 FACS Machine Can Sort Chromosomes

FACS machines can separate fluorescently labeled chromosomes from unlabeled ones. Liquid carrying a mixture of labeled and unlabeled chromosomes passes by a laser, which excites the fluorescent tags. Whenever the photo-detector detects fluorescence, the controller module directs that drop into the test tube on the left. When no fluorescence is emitted, the controller module directs the drop into the test tube on the right. This sorting procedure allows the separation of fluorescently labeled particles from unlabeled ones.

chromosomes ( labelled ^m unlabelled)

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