Gene probe

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Gene probes are poly- or oligonucleotides (mostly single-stranded or double-stranded DNA , more rarely RNA ) that have a complementary base sequence to the gene being sought and can attach to the appropriate DNA sequence of an (immobilized) DNA. The stability of the attachment depends on the conformity of the DNA sequences, the GC content and the length of the gene probes or the number of hybridized bases. All sequences that are not perfectly homologous can be separated again by intensive washing in order to only get signals in the case of exact matches.

DNA probes are generated by phosphoramidite synthesis , random priming , polymerase chain reaction (PCR) , nick translation , tailing or phosphorylation . RNA probes, also called riboprobe, are produced by in vitro transcription .

Gene probes are linked to marker molecules for the purpose of tracking or detection. For direct detection with radioactive isotopes ( 32 P, 35 S) or fluorescent dyes , for indirect detection via middle molecules, e.g. biotin via streptavidin or antibodies for digoxigenin . Depending on the further detection method, either (fluorescent) dyes or enzymes are coupled to the “middle molecules”. Frequently used enzymes for this are alkaline phosphatase or peroxidase .

Gene probes are detected either directly by detecting the ionizing radiation ( autoradiography or scintillation), or the corresponding dyes (fluorescence microscopy, photometry) or indirectly by enzyme substrate reaction, in which either chemiluminescence , dye change or dye precipitation is generated. The detection of the marker molecules (direct or indirect) counts as detection of the specific gene probe and thus also of the DNA or RNA to be analyzed with which the probe has hybridized.

application

Gene probes are used in genome analysis and expression analysis . Typical applications in genome analysis are the Southern blot and fluorescence in situ hybridization for chromosome analysis . Examples of application in expression analysis are the Northern blot , the RNase protection assay and in situ hybridization for the detection of mRNA.

See also

literature