By Thomas Liehr
This FISH program consultant presents an outline of the foundations and the elemental recommendations of fluorescence in situ hybridization (FISH) and primed in situ hybridization (PRINS), that are effectively used to check many features of genomic habit and adjustments. In 36 chapters, contributed by way of foreign specialists of their specific box, the these days a number of methods and purposes of the robust options are offered and exact protocols are given. defined listed below are tools utilizing numerous phone forms and tissues in addition to various organisms, similar to mammalians, bugs, vegetation and microorganisms. Multicolor FISH systems and specified functions corresponding to the characterization of marker chromosomes, breakpoints, cryptic aberrations, nuclear structure and epigenetic adjustments, in addition to (array-based) comparative genomic hybridization reviews are provided. total, the means of selection is brought for unmarried mobile research in human genetics, microbiology, animal and plant sciences.
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Extra info for Fluorescence In Situ Hybridization (FISH) - Application Guide
12. 13. 14. 15. 16. 17. T. Liehr, F. Pellestor different probes on the same slide using smaller coverslips. The amount of probe/probe-solution must be reduced according to the coverslip size. Incubate slides for 1–3 nights at 37°C in a humid chamber. 2% Tween (RT, 100 ml coplin jar). Go on with step 8 (smaller and/or more sensible probes) or 9 (larger and/or more stable probes). Post-wash the slides for 3 × 5 min in formamide solution (45°C) followed by 3 × 5 min in 2× SSC (37°C) in a 100 ml coplin jar, with gentle agitation.
4. Pellet the DNA by centrifugation at 15,000 rpm for 15 min, discard the supernatant, and dry the DNA pellet at room temperature (RT) or using a speed vac. 5. Dissolve the pellet from step 4 in 20 ml of hybridization buffer, vortex, and spin down. 6. Denature the probe solution at 75°C for 5 min, and do a pre-hybridization step at 37°C for 30 min if repetitive probe is not used. 2 Slide Pretreatment In a conventional FISH approach, pretreatment of the slides with pepsin followed by postfixation with formalin buffer is required to reduce the background.
The primers are annealed in situ to their denatured complementary DNA target sequences, and then extended by a Taq DNA polymerase in the presence of free nucleotides. The visualization of the generated fragments is enabled by the incorporation of one labeled nucleotide. The principle of PRINS is as follows (see also Fig. 2): – Fix the target DNA onto a clean microscope slide. Chromosome spreads, nuclei, extended chromatids or tissue sections can be used in PRINS reactions. – Prepare the PRINS reaction mixture incorporating the target-specific primer, the Taq DNA polymerase and its buffer, and the nucleotide mixture, including a labeled dUTP.
Fluorescence In Situ Hybridization (FISH) - Application Guide by Thomas Liehr