Fluorescence In Situ Hybridization

2023-10-30 Hits(191)

1. Principle of Fluorescence In Situ Hybridization


Fluorescent molecules can be deposited in chromatin at the sites of specific DNA sequences by use of fluorescence in situ hybridization (FISH). The technique is a simple one. DNA or RNA sequences are first labeled with reporter molecules. The probe and the target chromosomes or nuclei are denatured. Complementary sequences in the probe and target are then allowed to reanneal. After washing and incubation in fluorescently labeled affinity reagents, a discrete fluorescent signal is visible at the site of probe hybridization.

Using haptens such as biotin, digoxin indirectly labeled or fluorescein directly labeled known nucleic acid molecules as probes, the probes are hybridized with the target sequence double-stranded DNA after denaturation, and the complementary heterologous single-stranded DNA molecules are annealized at appropriate temperature and ionic strength to form stable heterologous double-stranded DNA, and the haptens are displayed by fluorescently labeled avidin or anti-digoxin antibodies. The hybridization signal was observed by fluorescence microscope.


2. Basic process of FISH


2.1 Chromatin Preparation and Denaturation

Cells are hypotonically swollen and fixed on slides by procedures developed for conventional banding analyses. Slides are incubated briefly at 70°C in a solution containing 70% formamide to melt the DNA into single strands, and are then fixed in cold ethanol to reduce strand reannealing before probe is added.


2.2 Probe Preparation

Probes are labeled with reporter molecules and broken into 200~400bp fragments, a size that maximizes specific hybridization and decreases background fluorescence. Although probes may be directly conjugated with fluorescent molecules 2, the most widespread approach is to label probes with reporter molecules that, after hybridization, bind fluorescent affinity reagents. Typical reporter molecules include biotin, digoxigenin, dinitrophenyl (DNP). amino acetvlfluorene (AAF), mercury and sulfonate. The first three of these reporter molecules are incorporated as labeled nucleotides using nick translation. Alternatively, probes can be labeled by PCR (polymerase chain reaction) amplification between known priming sequences or by RNA transcription from appropriate vectors in the presence of labeled nucleotides. AAE mercury and sulfonate are attached to DNA through chemical reactions. Size reduction is accomplished by nick translation itself or by sonication after chemical modification.


2.3 Hybridization

Labeled probes are mixed in a hybridization buffer containing formamide, salt and dextran sulfate. For repetitive probes, blocking DNA (e.g. sonicatcd herring testes DNA) is added to suppress nonspecific, binding of probe to chromatin and glass. After denaturation, the mixture is applied directly to slides. Incubation times as short as 15 min at 37°C are sufficient to detect chromosome-specific repetitive sequences. For unique sequence labeling using large insert probes, prehybridization of the probe mixture in excess unlabeled genomic or Cot1 DNA for up to an hour may be necessary to reduce repetitive sequence binding to the target. Hybridization between target and probe then continues overnight at 37°C.


2.4 Fluorescent Labeling

After washing to remove mismatched or unhybridized probe molecules, slides are incubated in immunofluorescent reagents to produce a fluorescent signal at the sites of probe hybridization Fluorochrome conjugated avidin or anti-biotin antibodies are used to label biotinylated probes, whereas mecury-labeled probes are detected by incubation with a ligand carrying both a sulfhydryl group and the hapten DNP. Digoxigenin, DNP, AAF or sulfonate are labeled with specific antibodies followed by fluorescently labeled anti-immunoglobulins; the most commonly used fluorescent labels are fluorescein isothiocyanate (FITC), rhodamine and Texas Red, although a variety of other fluorescent tags with spectra ranging from blue after UV excitation (AMCA) to infrared after red light excitation (Cy5) are also available for multicolor analysis.


2.5 Banding

Chromosomes can be banded after hybridization, and although the bands are crude they are sufficient to identify chromosomes and regionally localize probes. Banding methods include actinomycin/DAPI staining for G-bands13;growth in bromodeoxyuridine, Hoechst staining and UV irradiation for R-bands14; or FISH of Alu or LINE repetitive sequences for R- or G-bands, respectively. Hybridization of the repetitive L1 probe is used to produce characteristic bands in mouse chromosomes.


3. Advantages of FISH


(1) Fluorescent reagents and probes are economical and safe;

(2) The probe is stable and can be used within a year after one labeling;

(3) The experiment period is short, the results can be obtained quickly, the specificity is good, and the localization is accurate;

(4) FISH can locate DNA sequences up to 1kb in length, and its sensitivity is comparable to that of radioactive probes;

(5) Multicolor FISH can detect multiple sequences simultaneously by displaying different colors in the same nucleus;

(6) FISH can either show changes in the number or structure of metaphase chromosomes on a slide or the structure of interphase chromosome DNA in a suspension.


KMD Bioscience can provide one-stop FISH technical services, including probe design, sample processing, hybridization detection, gene expression research, data analysis, etc., to achieve the standardization and standardization of detection procedures, improve the accuracy of experimental results, reduce false positive and false negative, and ensure to provide customers with high-quality FISH services.



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