3. Application: M-FISH labeling schemes
3.1 Custom-made vs. commercial-made nucleotides
Commercial nucleotides (Fig.10, m1-k1) and custom-prepared nucleotides (Fig. 10, m2-k2) were used for side-by-side M-FISH comparisons. Hybridization results show comparable M-FISH analyses. Same amounts/volumes of FITC, BIO and Cy5 labeled probes were used. The amount of labeled probe was increased by 30% for DIG (to compensate for phenol extraction losses) and 60% for Cy3 (to compensate for a less optimal batch of dye). The shorter exposure times for the M-FISH with commercial nucleotides, indicated that purified nucleotides labeled the DNA better. This was expected, considering that the same volume of any nucleotide contained 100% labeled dUTP for the commercial source but only 50-60% for the custom-made ones. Because of this, to achieve similar labeling efficiency, 2/3 of dTTP in a reaction needs to be replaced by custom-modified nucleotides (compared to 1/3 for commercial nucleotides).
Fig. 10.m1-k1and m2-k2. Comparison of metaphases and karyotypes of M-FISH analyses with commercial labeled nucleotides (m1-k1) and custom-synthesized labeled nucleotides (m2-k2). Amount of each probe used is shown in Table 2. The commercial nucleotides were: biotin-11/16-dUTP (Enzo Diagnostics or Boehringer Mannheim), FITC-12-dUTP, digoxigenin-11-dUTP (Boehringer Mannheim), Cy3-dUTP and Cy5-dUTP (Amersham-Pharmacia). The custom-made dUTP were conjugated to the same dyes/haptenes. BIO was detected with Avidin-Cy3.5 and DIG with Cy5.5. Both approaches yielded analyzable results. Exposure times were shorter for the M-FISH with commercial nucleotides, but S/N values were comparable (Table 2).
3.2. Fluorescent nucleotides vs. haptene nucleotides
M-FISH analysis using only fluorescent-labeled nucleotides (DEAC, FITC, R6G, TxR, Cy5) was compared with M-FISH using only haptene-labeled nucleotides (DIG, BIO, DNP, TAMRA and FITC ? here the fluorophores were used as haptenes and were detected with specific antibodies). Fluorescence filters appropriate for the use of all dyes were commercially obtained (Chroma Technologies, Table 3). When compared with the M-FISH using fluorescent labeled nucleotides only, results show that the use of haptenes increases the brightness of the signal (2-3 times reduced exposures) but decreases the signal to noise ratio (S/N, Table 2). The quality of labeling of any one chromosome depends on the quality of the painting probe itself, the amount of labeled probe used, the efficiency of the labeling reaction, the dye/haptene used and the overall amount of DNA in the same hybridization. For example, when twice as much volumes of each labeled library were used ("m1" vs. "m5", Table 2), results showed increased signal quality for the fluorescent-labeled libraries (FITC, Cy3, Cy5) but unchanged (BIO/Cy3.5) or decreased signal quality (DIG/Cy5.5) for the haptene-labeled libraries (also between m3 and m4). The decrease in S/N for the haptene-labeled probes may by due to the increased background hybridization of the probe, detectable by the labeled-antibody. At the same time, brightness of haptene-labeled probes is higher, with exposures up to 5 times shorter. The use of antibodies ("m4-k4", Fig.11) also decreases the smoothness of the hybridization signal, which appears more "dotty" overall. Choosing the appropriate fluors and haptenes for FISH should depend on the sensitivity of imaging equipment, probe size (single copy or paint probes) and the cost of each dye or haptene. In our hands, the most convenient nucleotide combinations for M-FISH uses DEAC, FITC, R6G, TxR, and BIO (detected with Avidin-Cy5). In M-FISH, imaging time of any channel/dye needs to be between 1/3 and 3x of the value for the two adjacent channels. If the FISH signal in any one channel is weak, thus requiring 5x-10x longer exposure times compared to an immediately adjacent channel, there is the possibility of signal "leaking" from the bright channel into the weak channel, thus interfering with the analysis accuracy and S/N quality.
Fig 11. m3-k3show results of M-FISH using custom-synthesized, fluorescent-labeled nucleotides only. The fluorophores used were: DEAC, FITC, R6G, TxR and Cy5 (Table 2). Results indicate that coumarine and rhodamine derivatives can be successfully used to replace the more expensive cyanine dyes. Cy5-dUTP labeled libraries can be labeled with BIO-dUTP and detected with Avidin-Cy5, a cheaper and more robust alternative (not shown). m4-k4 show results of M-FISH using dUTP custom-labeled with five different "haptenes". The detection scheme included: avidin-AMCA (BIO detection); mouse-antiDIG and horse antimouse Cy5.5 (DIG detection); rat antiDNP and donkey antirat Cy5 (DNP detection); goat antiFITC and donkey antigoat FITC (FITC detection); rabbit antirhodamine and donkey antirabbit Cy3 (rhodamine detection). All antibodies were stored as 1 mg/ml stock solutions and were used at 1:100 dilutions in 4xSSC, 5-10 minutes at 37 C.