Molecular Hybridization of Nucleic Acids
One of the most powerful methods of genetic analysis is hybridization technique. It develops high sensitivity, allowing the discovery of near to 10-10 g of investigated nucleic acid. Hybridization method is based on complementary interaction of single-stranded DNA or RNA molecules resulting in specific formation of double-stranded complex.
At the initial step of hybridization test (e.g., dot or spot hybridization) the sample, containing unknown nucleic acid sequence is heated or treated by alkali to produce single-stranded DNA molecules (DNA melting).
Then single-stranded DNA is adsorbed on some solid phase (e.g., nitrocellulose paper sheet).
Afterwards the sample is treated by specific hybridization probe.
The probe is the known short sequence of one-stranded DNA molecule, complementary to investigated nucleic acid sequence and labeled with highly sensitive tag – fluorescent or chromogenic dye, or radiochemical label.
The labeling with fluorescent dye is the most common now. Also radioactive 32P phosphate isotope can be applied as radiochemical tag.
If the investigated specimen contains the nucleic acid of interest, the probe will bind to its complementary sequence. After thorough wash the specimen fluorescence or radioactivity is analyzed. Positive samples demonstrate the increased levels of activity.
Hybridization probes are in routine use for investigation of complex mixture of nucleic acids. Specific DNA sequences separated in agarose gel can be detected by Southern blots, a method that uses hybridization of DNA to DNA.
Likewise, hybridization of probe to RNA by Northern blots can evaluate RNA synthesis.
Hybridization in situ is employed to discover microbial DNA and RNA in cells and tissues of different origin. In these cases frozen microscopic sections of tissue samples treated by fluorescent probes are tested.
The most advanced methods of nucleic acid hybridization exploit DNA microarray technologies. They are based on detection of mass parallel hybridization of thousands of nucleic acid probes and clinical samples. In this case an array of DNA probes taken in micro-quantities is absorbed on glass or plastic solid phase thereby producing DNA biochip. It is next treated with the mass of clinical samples containing microbial nucleic acid with subsequent fluorescent registering of dot hybridization.
This technology allows parallel testing of thousands of clinical specimens for specific microbial DNA or RNA as well as simultaneous detection of all of microbial pathogens present in clinical sample