Covers hybridization as the pairing of complementary nucleic acid strands, going through denaturation, annealing, stringent washing and detection, then probes and their labels in a table. It describes in situ hybridization, including FISH examples such as trisomy 21, 22q11.2 deletion, BCR–ABL1 and HER2 amplification, and blot methods in a comparison table. A further table sets ISH against immunohistochemistry. The second half introduces cell culture: cell lines versus primary cells, culture components, basic steps, applications and microscopy techniques.
Topic 6 of 127 in the MU Sofia syllabus for Cytology (Dental Medicine, Year 1). The free AceMed note covers it in 6 sections, about 1,365 words (11 min of reading), with 16 flashcards.
In this note
Introduction
Principle
Probes and labels
In situ hybridization (ISH)
Membrane (blot) hybridization
ISH compared with immunohistochemistry
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.
Continue reading free
The whole note, all 6 sections, is free with an AceMed account, with its 16 flashcards. Premium adds flashcard recall, highlights and Ask AI.
