Short DNA probes
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Plasmids

Short DNA plasmid probes (from 100 to 3000 bp) can be labeled using either nick translation or PCR. For very short probes, PCR is probably more efficient, whereas for longer fragments both labeling technique will work. This is illustrated in Fig. 9f and 9g, in which a 3kb plasmid probe was labeled using either PCR with vector primers (Fig. 9f) or nick translation (Fig. 9g). Labeling and hybridization worked well with both procedures, but the FISH signal appeared somewhat stronger when using the PCR-labeled probe. However, as the DNase treatment of the PCR fragments was minimal and the labeled fragments remained somewhat larger (500-2000 bp), there was a relatively large amount of background on the slide after hybridization (Fig. 9f). By comparison, the nick translated DNA fragments yielded a much cleaner hybridization. This underscores again the importance of using labeled DNA fragments around 100-300 bp average.

PCR products

FISH can be performed with unique, very short DNA fragments (primarily PCR products). In Fig. 9h, two loci on chromosome Y (sY14 = 475bp on Yp and sY81 = 206bp on Yq) were amplified and labeled with DIG by PCR. The products were mixed together and hybridized onto normal male chromosomes. In some metaphases, two pairs of signals could be detected, the Yp ones (sY14) usually brighter by comparison. However, given the overall background on the slide, it would have been difficult to locate the signals without knowing their position. Many metaphases showed only one signal and signals were often not paired.

The reason for this variability is probably the reduced size of the labeled DNA fragments. The success of their hybridization depends on the chance that the corresponding DNA sequence is denatured and accessible for hybridization not only in every chromosome but also in each chromatid of the same chromosome. Denaturing is a more or less random process and the availability of any particular DNA stretch to hybridization also depends on the spatial position of the target sequence (whether it is towards the outside or is buried inside the chromatin). Therefore, hybridization of short sequences will result in double FISH signals (both chromatids) only occasionally.

By comparison, hybridization of a larger probe (a 200kb BAC) will result in a hybridization signal on almost every chromatid of every chromosome available on the slide. The labeled DNA fragments hybridizing to the target loci on the two chromosomes of the same metaphase are probably from different parts of the BAC. The reason is that on every chromatid, only random parts of the 200 kb target DNA will be denatured and available to hybridization with the labeled DNA probe fragments.