Explains how hybridization pairs a labelled probe with its complementary nucleic acid sequence, covering denaturation, annealing, stringent washing and detection. Describes probe types and three labelling systems, then in situ hybridization with FISH examples such as trisomy 21, BCR–ABL1 and HER2, before membrane methods (Southern, Northern blots, microarrays). Ends with a comparison table against immunohistochemistry.
Topic 7 of 97 in the MU Sofia syllabus for Cytology (Medicine, Year 1). The free AceMed note covers it in 9 sections, about 1,177 words (10 min of reading), with 13 flashcards.
In this note
Introduction
Principle
Probes and labels
In situ hybridization (ISH)
Membrane (blot) hybridization
ISH compared with immunohistochemistry
Microscopy. Types of microscopes.
Other Methods
Summary of Key Points
Introduction
Hybridization is the pairing of two single strands of nucleic acid whose base sequences are complementary. Histology and diagnostics use it to find one particular DNA or RNA sequence among all the others. A probe of known sequence carries a label and binds only where its partner sequence is, and the label shows where that is. Cell cultures have a note of their own.
Principle
Bases pair through hydrogen bonds: A–T in DNA (A–U in RNA) and G–C. A G–C pair has three hydrogen bonds and an A–T pair has two, so G–C-rich stretches hold together more strongly.
Denaturation: heat (about 90–95 °C) or chemicals such as formamide or alkali separate the double helix into single strands.
Annealing (hybridization): as the mixture cools, the probe finds and pairs with its complementary sequence. DNA can pair with DNA or with RNA, and RNA with RNA.
Washing removes probe that is unbound or only loosely bound to near-matches. The stringency of the washing (higher temperature, lower salt, formamide) decides how exact a match must be to survive.
Detection of the label shows where the probe has bound.
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