Denaturation Mapping

Denaturation of the double-helical structure of DNA is an essential first step in many of the most common methods for investigating chromosomal structure. The method relies on the unique order of bases in DNA and the capacity of two separate complementary strands to recognize each other through the process of hybridization. Denaturation mapping can be done using either sections of tissue or chromosomal preparations. The sample slide to be analyzed is heated to denature the double helix into two separate DNA strands. Rapid cooling of the preparation in the presence of formaldehyde keeps the DNA strands separated. The cloned gene or sequence of interest is prepared as a DNA probe in single-stranded form with a label attached. This label could be either a source of radioactivity, or an epitope recognized by an antibody (often using streptavidin as an antigen). A change in salt concentration and temperature facilitates hybridization between complementary DNA strands. The probe and chromosomal sequences compete with each other, but a sufficient excess of probe can be added so that some of the probe hybridizes to the specific site on a chromosome.

When initially developed by Pardue and Gall in 1970, the probe was labeled with the radioactive hydrogen isotope tritium (1). More recently the use of antibodies has become popular (2, 3). The antibodies are usually linked to an enzyme that acts on an appropriate substrate to generate a visible signal. This could either be a colored precipitate on the chromosome, that is detectable under a light microscope, or the antibody could be linked to a fluorochrome that is visualized by a fluorescence microscope. This latter technique is known as fluorescent in situ hybridization (FISH). Now modifications of this technique allow analysis under the electron microscope for increased resolution or in the confocal microscope that builds up three-dimensional images by optical sectioning.

A number of specialized methods have acquired their own acronyms. These include fluorescence in situ hybridization (FISH) as described; genomic in situ hybridization (GISH); whole chromosome painting (WCP); and primed in situ labeling (PRINS). GISH is a method for detecting species–specific chromosomes when the entire genomic DNA is labeled and hybridized to whole chromosomal spreads. It is useful in analyzing of cell fusion hybrids and in evolutionary studies. In WCP, DNA from a single chromosome is labeled and prehybridized with repetitive DNA to stop cross-reaction with other chromosomes from the same organism. WCP probes are available commercially for all 22 autosomes and the sex chromosomes in humans. In PRINS, the polymerase chain reaction (PCR) is used to label sequences on the actual chromosome or tissue. There are many other adaptations of these important methodologies.

References

1. M. L. Pardue and J. Gall (1970) Science 168, 1356–1358. 

2. J. Leary, D. Brigati, and D. C. Ward (1983) Proc. Natl. Acad. Sci. USA 80, 4045–4049.

3. P. Lichter et al. (1990) Science 247, 64–68.

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مواضيع ذات صلة

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Coupling Translation to Termination

trp Multiple Secondary Structures in trp Leader RNA

Early Explorations of the Hypersynthesis

The Serendipitous Discovery of trp Enzyme Hypersynthesis

Rapid Induction Capabilities of the trp Operon

The Aromatic Amino Acid Synthetic Pathway and its Regulation

How AraC Protein Loops and Unloops

DNA Looping and Repression of araBAD

Binding Sites of the ara Regulatory Proteins

Endoplasmic Reticulum-Associated Degradation and the Unfolded Protein Response

Protein Folding and Modifications in the Endoplasmic Reticulum