3-1.3.6 Hybridization-Based Techniques

Hybridization-Based Techniques for nucleic acid detection has higher resolution (down to the actual nucleotide sequence) and utilizes "probe sequence" for DNA or RNA, and when it finds its intended target, binds to it by hybridization process. Since the "probe" is attached to a label such as a fluorescent chemical isotope, the bound sequence can thus be visualized. This is the principle of Southern Blotting method, as well as its variants, and requires the target nucleic acid sequence to undergo separation by agarose-electrophoresis, transferred to an appropriate membrane (typically nitrocellulose), and then treated with a solution containing the labeled probe (fluorescent or colorimetric).

Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence. Since the probe specifically binds to its target, the membrane can be documented and analyzed so that the bound target sequences can be identified and studied. The detailed methodology is described in later chapter (Module-4Lecture 3).

FISH (Fluoroscence in-situ hybridization) for visualization of nucleic acids developed as an alternative to older methods that used radio labeled probes (Gall and Pardue, 1969). Several drawbacks of isotopic, radiolabeled hybridization stimulated the development of novel techniques like FISH. In the year 1980, RNA was first directly labeled on the 3' end with fluorophore was used as a probe for specific DNA sequences (Bauman et al., 1980). Enzymatic incorporation of fluorophore-modified bases throughout the probe has been widely used for the preparation of fluorescent probes. The use of amino-allyl modified bases (Langer et al., 1981), which could later be conjugated to any sort of hapten or fluorophores, was critical for the development of in situ technologies because it allowed production of an array of low-noise probes by simple chemistry. Methods of indirect detection result into higher magnitude signal output by the use of secondary reporter molecules that bind to the hybridization probes.

In the early 1980s, assays like nick-translated, biotinylated probes, and secondary detection

by streptavidin conjugates were used for detection of DNA (Manuelidis et al ., 1982) and mRNA (Singer and Ward, 1982) targets. Later, advanced labeling of synthetic, single-stranded DNA probes allowed the chemical preparation of hybridization probes carrying enough

fluorescent molecules to allow direct detection (Kislauskis et al ., 1993). At this current era, based on these reaction themes of indirect and direct labeling have since been introduced, a wide spectrum of nucleic acid detection schemes are available.

Whereas the initial development of FISH involved expansion of the types of probe and number of detectable targets, the outlook for future development of fluorescence techniques will include expansion of the subjects of investigation like in clinical, diagnostics, forensic applications.

Other detection methods include amplification of target region by polymerase chain reaction and various forms of chemiluminescent, fluorescent or radioactive detection method. Specific technical or sample requirements are present for each method, and the original purpose for detection the nucleic acid will determine which of the methods is most suitable.