Cost reduction of FISH with commercial probes
The "lower stringency" approach
Although commercial probes are provided as kits with ready-to-use solutions, they should not be regarded as unchangeable entities. All DNA probes behave like any other DNA probes, and protocols can be modified and improved. Moreover, probes from different kits can be combined.
For example, a cosmid or BAC probe (usually washed in 0.2xSSC at 65° C post- hybridization) can be combined with a painting probe (usually washed in 2xSSC at 37° C). In such cases, the second posthybridization wash (in SSC) will be performed at the temperature used for the painting probe (less stringent wash). If background from the BAC is a concern, 10-15 ?g competitor DNA can be added to the cosmid-paint probe mixture prior to hybridization, followed by ethanol precipitation, resuspension in hybridization buffer, denaturing and hybridization. The extra Cot1-DNA will block repetitive sequences better.
The "low amount of probe" approach.
Commercial probes kits provide sufficiently large amounts of labeled probe, so as to allow hybridization even in less optimal conditions. If the FISH procedure is efficiently, DNA probes purchased in kits can be used for 3-5 times more hybridizations than indicated, thus reducing the cost for each hybridization. This was tested in this laboratory numerous times. For example, in Fig. 6, three different commercial probe kits were used. However, for each hybridization 1/4 or 1/5 of the recommended amount of probe was used (2 ul of the provided probe was mixed with 8-9 ul hybridization buffer). Simultaneous or separate denaturing worked just as well. Moreover, as shown in Fig. 6, good hybridization results were obtained after only 3-5 hours hybridization. Overnight hybridizations did not improve the signals in any ways and did not seem necessary. This shows that, even the reduced amount of each probe used, could be further decreased.
The "replacement" approach
If fluorescence-antibodies are necessary, they can be purchsed either non-labeled or labeled (but in higher amounts) from other vendors. Non-labeled antibodies can be easily labeled in any laboratory. Besides, the fluorophore used for any probe detection can be replaced with any other one at will, the only limitation being the number of filters and the quality of the filters with which a fluorescence microscope is equipped. A summary of some of the fluorophores used in this laboratory is presented here or here (Please note that this list, although it covers the major groups of fluorophores, is by no means complete. There are many other fluors available comercially).
Mixing of multiple commercial and custom probes
When purchasing them from vendors, commercial probes usually arrive in hybridization buffer, denatured, re-annealed and combined with competitor DNA. If probes from three or four kits or sources are to be used together, in the same hybridization, the final volume may be higher than the 10-12 ?l accomodated under a standard 22x22 mm coverslip. To solve this problem, all probes can be co-precipitated together. That the probes may have been previously denatured has no importance. For example, if the total volume of the mixed probes is 25-30 ?l, they can be resuspended in 5-10 volumes of TE or water (to approximately 200 ?l) followed by ethanol precipitation. If desired, 15-20 ?l competitor DNA can also be added. The pellet is resuspended in 10-12 ?l hybridization buffer and used. An example of this approach is shown in Fig. 10d. A commercial, dual-color probe (red=rhodamine and green=FITC) was mixed and used together with a biotinylated commercial paint probe for a chromosome in group F. As recommended by the vendors, 10 ?l of each probe were mixed in the same vial, 20 ?g Cot 1 DNA was added, and the DNA was precipitated, resuspended in hybridization buffer and hybridized. The biotinylated probe was detected with a third color (avidin Cy5, available in our laboratory), and the chromosomes were counterstained with DAPI. All three probes could be visualized, although they were not used according to the protocols provided by the two vendors.
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Last modified on: Feb12, 2001