This webpage was created by Bernard Blommel & Alan Handler
1. OVERVIEW OF FLUORESCENCE IN SITU HYBRIDIZATION
Fluorescence in situ Hybridization (FISH) is a microbial method that allows for the detection of whole-bacterial cells and the analysis of chromosomes via the labeling of specific nucleic acids with fluorescently labeled oligonucleotide probes. Basically, the FISH method uses fluorescent molecules to vividly paint genes or chromosomes so that they can be detected and identified. First, short sequences of single-stranded DNA, called probes, are prepared. These probes hybridize, or bind, to complementary nucleic acids and, because they are labeled with fluorescent tags, allow researchers to see the location of those sequences of DNA. When the results are viewed, usually with a scanning laser microscope, they can be visually stunning. Unlike most other techniques used to study microorganisms, which require that the cells be actively dividing, FISH can also be performed on nondividing cells, making it a highly versatile procedure. The FISH method involves four steps: fixation, hybridization, washing, and detection.
2. TYPES OF PROBES
Scientists use three different types of FISH probes, each of which has a different application:
Locus specific probes hybridize to a particular region of a chromosome. This type of probe is useful when scientists have isolated a small portion of a gene and wish to determine which chromosome that gene is located on. They prepare a probe from the piece of the gene and observe which chromosome the probe hybridizes to.
Alphoid or centromeric repeat probes are generated from repetitive sequences found at the centromeres of chromosomes. Because each chromosome can be painted in a different color, researchers use this technique to determine whether an individual has the correct number of chromosomes or, for example, whether a person has an extra copy of a chromosome.
Whole chromosome probes are actually collections of smaller probes, each of which hybridizes to a different sequence along the length of the same chromosome. Using these libraries of probes, scientists are able to paint an entire chromosome and generate a spectral karyotype. This full color image of the chromosomes allows scientists to distinguish between the chromosomes based on their colors, rather than based on their dark and light banding patterns, viewed in black and white through traditional karyotyping. Whole chromosome probes are particularly useful for examining chromosomal abnormalities, for example, when a piece of one chromosome is attached to the end of another.
3. PROS AND CONS
Fluorescence in situ hybridization has many advantages over conventional laboratory cultivation techniques. This method allows for the in situ localization and the study of spatial organization of cells as they occur in their natural habitat. This is great for studying the actual composition of a natural microbial community. FISH also allows for the detection of one to three orders of magnitude more cells in samples. On top of that, this method requires no cultivation of cells before analysis. Another added advantage is that for FISH, cells need not be alive. The intensity of the fluorescence is a direct measure for the activity of the cells themselves. Inactive cells can be recognized by their low intensity fluorescence.
There are, of course, some disadvantages of fluorescence in situ hybridization. The process of preparing probes is complex due to the fact that it is necessary to tailor the probes to identify specific sequences of DNA. Also, it is difficult to count total numbers in probe-stained clusters of cells.
Amann, R.I. 1987. In situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes, p. 1-15. In A.D.L. Akkerman, J.D. van Elsas, and F.J. de Bruijn (ed.), Molecular microbial ecology manual. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Okabe, Satoshi, et al. 1999. In situ analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes, In Applied and Environmental Microbiology. 65:3182-3190.
5. LINKS TO OTHER SITES RELATED TO FLUORESCENCE IN SITU HYBRIDIZATION
Fluorescence in situ hybridization (FISH) of C3 and 18S rDNA to horse chromosomes
| HOME PAGE | HONOR CODE | SYLLABUS | CHAPTERS | CYCLES |
| MICROBES | APPLICATIONS | PROJECT | LINKS |