By K H Andy Choo
Thirty-one designated, useful chapters many via the originators themselves current easy-to-follow protocols of in situ hybridization ideas for mapping DNA sequences onto chromosomes, quantitation/localization of RNA in tissues, and detection of virus nucleic acids. The equipment serve molecular biologists in so much parts of simple and utilized study, and in each type of organism starting from viruses to Drosophila to people. The professional members supply step by step directions for the practise of probes for DNA, teleomeric repeats, cDNA sequences, small man made oligomers, complete cosmids, mega-size YACs, and chromosome-specific paints from somatic phone hybrids and flow-sorted chromosomes. in addition they current radioactive and nonradioactive probe labeling methods with using nick translation, random priming, multicolor labeling, PRINS labeling of DNA and RNA, and PCR in situ hybridization. colour illustrations increase the textual content. Troubleshooting suggestions, alternative routes of doing issues, and informative factors of why definite steps are performed -- aids no longer often present in usual magazine recipes -- warrantly an important distinction within the consequence of experiments.
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Additional resources for In Situ Hybridization Protocols (Methods in Molecular Biology Vol 33)
Gently remove air bubbles and seal the edges with rubber cement to minimize evaporation 2. Hybridize at 37°C in a moist chamber contammg absorbent paper soaked in water. For repetitive probes, hybridize for a few hours to overnight. 6. 1. wash conditions Wash are required depending on the type of probe used. FOR COSMID, PHAGE, OR YAC PROBES CONTAINING REPETITIVE ELEMENTS 1 Prewarm wash solution A to 46°C in three plastic jars 2. With forceps, carefully remove the rubber cement from the slides Allow the covershps to float off in 2X SSC solution Wash the slides a few times by agitating 3 Wash slides m wash solution A by gently agitating three times for 3 mm each.
It is often dtfficult, however, to precisely position the hybridization signals with respect to the conventional chromosome bands. Various approaches have been introduced, including G banding before or after FISH detection (2,3), cohybridization with Alu or L 1 probes to generate R or G bands (4), use of a combination of various fluorescent dyes, such as qumacrine, Hoechst 33258, or DAPI, with FISH detecting reagents (5,6), and triple staining with chromomycin, distamycin A, and DAPI (7,8). Unfortunately, these procedures are not widely used because they are time-consummg and require extensive imaging equipment or careful quality control to be reproducible.
Typically, we expose FITC signals for 4-5 s, then expose DAPI pattern on the same film for 1 s by double exposure. Two factors should be considered: First, the imaging of DAPI and FITC will not be perfectly aligned In order to get precise locahzatton, around 10 mitotic figures should be used for position determmation Second, when this FISH on DAPI-Banded Chromosomes 47 method is used, PI staining should be avoided, since the PI counterstainmg will reduce the DAPI banding differentiation after double exposure.