The development of vast array of laboratory methods and their applications provided great leaps in the ability of the researchers to discover new features and functions of macro-molecules. Most of them represent procedures for measuring or visualizing ever-smaller quantities or tinier features of molecules, or part of molecules. Especially when applied in combination, these methods have led to enormous advances in understanding the structural features of proteins and nucleic acids. New techniques have been regularly introduced and the sensitivity of older techniques greatly improved upon. The originators of several of those breakthrough methods were awarded Nobel Prizes. Basic principles of some of most important techniques invented and applied in molecular biology research are described in this chapter.
TopFluorescence In Situ Hybridization (Fish)
Fluorescence in situ hybridization (FISH) is a technique used to analyze chromosomes at the gene or DNA level. Both metaphase (dividing) and interphase (no-dividing) cells are used to identify structural abnormalities in the chromosomes through FISH. FISH is usually applied to cytogenetic preparations on microscopic slides, but it can be used on slides of formalin-fixed tissue, blood or bone marrow smears, and directly fixed cells or other nuclear isolates. The basic principle on which FISH works is that a dsDNA molecule can be denatured to form ssDNA, and a complimentary strand (other than the original complimentary strand) can bind to the ssDNA thus generated. Thus, a specific DNA sequence in the metaphase or interphase chromosomes can be identified by using a DNA probe having complimentary sequence. However both the chromosomal DNA sequence and the probe should be in single-stranded conformation, which can be achieved by heating them in a solution containing formamide. The advantages of FISH over ISH (in situ hybridization) are faster detection, higher resolution, sensitivity and speed.
The target DNA sequence and the probe get hybridized to form dsDNA under ideal conditions. An excess of the repetitive sequence DNA is added to the hybridization mixture to avoid non-specific binding. Depending on the nature of the probe and target DNA, hybridization process should be completed in about 2-18 hr at 370 C. Thereafter the excess non-bound probe is removed by washing the slides with formamide-saline citrate solutions. The probe should be previously labeled directly with a fluorescent tag so as to locate them on the target DNA. The probes can also be labeled indirectly first by joining with a haptan molecules (biotin or digoxigenin) and then binding the haptan with a fluorescent tag. The target DNA is counterstained with another fluorochrome of a complimentary color.
With the help of a fluorescent microscopy and specific filters it is possible to observe the flourochrome labeled probe bound to the target DNA. With the help of special filters it is possible to observe several flourochromes simultaneously. To increase the sensitivity of detection of the probes, digital cameras capable of detecting low light intensity along with computer imaging software are used. The FISH preparations tend to fade over time (photobleaching) and therefore should be stored in the dark. However it is possible to improve the longevity and documentation of the FISH preparations by application of anti-fad solution like phenylenediamine.
When total genomic DNA (consisting of the entire nuclear DNA of a species) is used as a probe in hybridization experiments to chromosomal DNA in situ, the technique is called GISH (genomic in situ hybridization). GISH permits characterization of the genome and chromosomes of hybrid plants, allopolyploid species, recombinant breeding lines, and phylogenetic relationship. Multicolor FISH (mFISH) using genomic DNA probes are a promising approach for simultaneously discriminating each genome in natural and artificial amphidiploid.
The normal FISH approaches are based on air-drying procedure of chromosome preparation, which leads to well spread metaphase chromosome preparations. But when this procedure is adopted to study spherical interphase nuclei, the nuclei becomes flattened, and thus may lead to questionable results. To overcome this problem, a procedure called suspension FISH (sFISH) was developed, which allows 3D analysis. In this technique suspended cells are placed in polished concave slides as the final step of the procedure, just before evaluation.
Three different types of probes used in FISH studies are as follows: