DNA: DNA in situ hybridization
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Procedures for in situ hybridization of labeled DNA probes to spread or sectioned chromosomes and nuclei (DNA) are described here. Double-target experiments are recommended for most applications, while multiple-target or reprobing of preparations are possible if more information is required. Specialized alternatives use Cell s in suspension or whole mount preparations as targets for hybridization. Detection and visualization of probe-target hybrids is described in Chapter 9 and procedures to detect RNA targets are covered in Chapter 10. Most experiments use long probes (from clones or genomic DNA, on average 100-500bp long after labeling). Labeled synthetic oligonucleotides (14-30bp long) are being used increasingly as probes, when salt and temperature conditions need to be adjusted. Oligonucleotides are also used for in situ primer extension and in situ PCR protocols, particularly to detect repetitive sequences
THE NORMAL DNA:DNA IN SITU HYBRIDIZATION PROCEDURE Use of one or more tested, labeled probes Use of checked slide or grid preparations carrying target sequences Pretreatment of the chromosome preparation prior to the hybridization to remove RNA, cytoplasm and other cellular material, and to make the cells and chromatin permeable to the probe Refixation of the material to help maintain chromosomal morphology, and prevent loss of material during subsequent steps Decision on hybridization stringencies and making of hybridization mixture Denaturation (making DNA single-stranded) of probe and chromosomes, before hybridization, normally overnight, when probe-target hybrids will form Stringent washes to remove unbound or loosely bound DNA probe Detection of hybridization sites and counterstaining of the chromosomes
During the in situ hybridization procedure it is important to work accurately and cleanly to ensure results are reproducible, to reduce the accumulation of dirt that causes background, and to reduce loss or damage to the material. All tools and containers must be clean, and solutions without fungal growth, although aseptic conditions are not necessary. The preparations on slides must not touch other slides (a frequent cause of scratching material), nor dry out or accumulate water when incubating and between steps. Wash solutions should cover slides totally and should be changed carefully to avoid strong turbulence. In the average experiment, it is convenient to handle eight slides at once: these fit easily into one staining jar for the washing steps. Fifteen or 20 slides can be handled if two staining jars are used or extra slides are placed diagonally in the jars (but it is easy to leave slides behind in the washes).
Most protocols involve an overnight hybridization step of about 16 h, sufficient time for most probes to find homologous sequences on the chromosomes. It also gives ample time on the first day to set up the experiment; the next day has enough time for the stringent washes and detection. Several one-day protocols are published using only 2-4 h of hybridization; they work well for repeated sequences, and primer-extension protocols require even less primer annealing time Special equipment Equipment required for multiple protocols is shown in the box.
