In situ DNA hybridization

In diagnostic clinical medicine, DNA identification has been applied to investigations of patient tissue specimens obtained during biopsies or autopsies. Because of imaginative adaptations of DNA extraction techniques, the DNA contained in slides and preserved tissue specimens can now be investigated for evidence of infectious or genetic diseases. For example, in situ DNA hybridization is an ultrasensitive technique in which specific DNA probes are applied directly to tissue embedded in paraffin. (Paraffin-embedded tissue specimens are used in microscopic studies. Such slides can be stored... 

Figure 14.12. Electrochemical impedance spectroscopy based in situ DNA sensing (A Nyquist plots and (B corresponding Bode plots of as grown, DNA probe against anthrax lethal factor (pLF -modified, and dsDNA-modified GaNNWs at different concentrations of LF targets (arrows indicate increasing concentration, in situ DNA hybridization detection. ...

L3. Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and Ward, D. C., Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80, 224-234 (1988). 

Figure 6.18 ImmunoRCA Assay. Top left A reporter Ab conjugated to an oligonucleotide binds to a test analyte captured on a solid surface by a covalent attachment or by a capture Ab. Top right DNA circle hybridizes to a complementary sequence in the oligonucleotide. Bottom left Resulting complex is washed to remove excess reagents, and the DNA tag is amplified by RCA. Bottom right Amplified product is labeled in situ by hybridization with fluor-labeled oligonucleotides. (From Schweitzer, B. et al, Proc. Natl. Acad. Sci. USA, 97, 10113-10119, 2000. With permission.)...

A diagnostic method using fluorescence labeled DNA probes to detect and quantify the number complementary chromosomal sequences on a cellular resolution. A related technique that also allows assessment of gene amplifications, but without precise quantification of copy numbers is the chromogenic in situ hybridization (CISH). Here, instead of a fluorescent dye an enzyme that can generate a colored precipitate in the tissue samples is coupled to the DNA probe. 

FISH. Fluorescent in-situ hybridization a method utilizing fluorescently labeled DNA probes to detect or confirm gene or chromosome abnormalities that are generally beyond the resolution of routine cytogenetics. 

A variation on this method, called fluorescent in situ hybridization (FISH), uses fluorescent-labeled DNA and RNA probes for detection and visualization of single cells by microscopy or flow cytometry.7 80 The FISH technique is popular because of its sensitivity and speed of visualization fluorescent dyes can be used to produce probes with different colors for simultaneous detection of several organisms.76,81,82... 

In situ hybridization Use of a DNA or RNA probe to detect the presence of the complementary DNA sequence in cloned bacterial or eukaryotic cells. 

Fluorescently labeled DNA probes can be used for detection, localization, or quantification of target DNA sequences. In situ hybridization mapping of genomic DNA sequences can be... 

The last section of this chapter includes in brief a procedure of McF adden (9) for in situ hybridization. In situ hybridization relies on the complementarity of the bases contained within DNA and RNA. In addition to the hybridization (reassociation) of complementary DNA strands, hybridization is possible between DNA and RNA strands that are complementary. Also, hybridization is possible between a synthetic sequence and a sequence of biological origin. In situ hybridization may be used to determine the location of a specific nucleic acid sequence within a cell. The procedure requires the use of a probe for the sequence of interest. The probe, in turn, must be complementary to the sequence of interest. The probe may be either single stranded or double stranded, DNA or RNA. There must exist a method by which to detect the probe. 

Molecular biology involves the study of the major macromolecules, DNA, RNA, and protein. The central dogma ofmolecular biology is illustrated in Fig. 2. The central dogma shows the relationship among the macromolecules in the processes of transcription and translation. Figure 2 also gives the relationship between immunoelectron microscopy and in situ hybridization. In situ hybridization allows one to localize a specific nucleic acid sequence. Immunoelectron microscopy is an essential component to the technique of in situ hybridization when applied at the EM level. 

The RNA molecules, ribosomal RNA (rRNA) and messenger RNA (mRNA) play key roles in the protein synthesis. The amount of RNA in individual cells or in a community may, therefore, be taken as an indicator of protein synthesis and, thus, microbial activity. The number of active cells can be detected by fluorescent in situ hybridization (FISH) (Amann et al. 1995). By this method, individual cells carrying high concentrations of rRNA, situated on ribosomes, are quantified by fluorescence microscopy. The amount of rRNA in a community can also be detected by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), where rRNA extracted from soil is detected by creating a DNA copy and separating by gel electrophoresis (Duineveld et al. 2001). 

Some fluorescent DNA stains can also be used for chromosome counterstaining, for detection of hybridized metaphase or interphase chromosomes in fluorescence in situ hybridization assays or for identifying apoptotic cells in cell populations (http //probes.invitrogen.com/handbook/sections/0806.html). For instance, Vybrant Apoptosis Assay Kit 4 (Molecular Probes) detects apoptosis on the basis of changes that occur in the permeability of cell membranes. This kit contains ready-to-use solutions of both YO-PRO-1 and propidium iodide nucleic acid stains. YO-PRO-1 stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide. 

Bentolila LA, Weiss S (2006) Single-step multicolor fluorescence in situ hybridization using semiconductor quantum dot-DNA conjugates. Cell Biochem Biophys 45 59-70... 

Advances in the fluorescence in situ hybridization (FISH) technique with the ability to label DNA probes with as many as six different spectrally distinct... 

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