BRIEF REPORT |
Increased Efficiency of Fluorescence In Situ Hybridization (FISH) Using the Microwave
Institut für Humangenetik und Anthropologie, Jena, Germany (AW,TL,UC), and Institut für Anatomie II, Jena, Germany (K-JH)
Correspondence to: Dr. Thomas Liehr, Institut für Humangenetik und Anthropologie, Postfach D-07740, Jena, Germany. E-mail: i8lith{at}mti.uni-jena.de
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Summary |
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Key Words: FISH microwave effectiveness
FLUORESCENCE IN SITU HYBRIDIZATION (FISH) is a technique well established in human cytogenetics (for review see Liehr et al. 2004; Liehr 2005
). The hybridization efficiency achieved using standard protocols is sufficient for routine applications in most cases. Nonetheless, there are reports about enhancing FISH effectiveness by use of microwave via pretreatment of tissue sections and subsequent hybridization (e.g., Coates et al. 1987
; Sibony et al. 1995
; Bull and Harnden 1999
; Kobayashi et al. 2000
). In this respect, microwave was also applied for decondensation and denaturation of DNA (for review see Ko et al. 2001
) and for enhanced FISH signals of repetitive probes (for review see Kitayama et al. 2000
). Even though microwave is widely used as well in immunohistochemistry, there are no studies on its mode of action.
Here we present a new microwave-FISH protocol to enhance FISH signals on chromosomes in which microwave pulses were applied during the DNA-DNA hybridization process. Using this approach, quick results or enhancement of weak signals can be achieved.
The described microwave-FISH protocol (see the following section) was tested in comparison to normal FISH without microwave (Liehr et al. 1995) using the BAC probe b35B4. This probe derives from chromosome 1q21 and contains 143 kb of nonrepetitive DNA; however, it produces three specific FISH signals in 1q21, 1p12, and 1p36.1 (Figure 1). A total of 123 kb of b35B4 sequence is present in at least five copies in 1q21, leading to a strong FISH signal there. In 1p36.1, 59 kb of b35B4 sequence are present twice in tandem order. Additionally, 21.5 kb of b35B4 sequence are located once in 1p12. This probe was chosen because its application of one FISH probe leads to three signals of different intensity (Figure 1) (Weise et al. 2005
).
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Furthermore, autoexposure times after one microwave beam with and without subsequent 10-min hybridization at 37C were compared. Because the autoexposure times showed no differences (data not shown), it can be speculated that microwave irradiation may act as a steric modifier, allowing the DNA probe to find the DNA target in a more efficient and faster way than in a normal FISH assay in which it depends on diffusion of probes to target.
In summary, the described microwave technique here is the first to enhance the efficiency of a single/low-copy DNA probe; all previous publications using microwave were dealing with centromeric probes (Kitayama et al. 2000; Ko et al. 2001
). The technique is applied routinely in our laboratory for subcentromere-specific, multicolor FISH probe sets (Starke et al. 2003
); in prenatal cases with small supernumerary marker chromosomes, this time-saving possibility was used obtain results within 2 hr. Moreover, we compared the dynamics of hybridization with and without application of microwave, demonstrating the efficiency of microwave irradiation especially in the first 100 min of hybridization. In practice, we recommend a treatment approximately 45 microwave beams at 600 W within 30 min to get a clear effect.
The used BAC DNA was isolated from Escherichia coli and adjusted to a final concentration of 100 ng/µl. Subsequently, 2 µl were used for degenerated oligonucleotid primersPCR amplification (amp-PCR: 50 µl volume; 30 cycles) and labeling (Texas Red/Biotin labeling-PCR: 20 µl; 20 cycles). Finally, the probe was dissolved in 30 µl hybridization solution (for PCR details, see Telenius et al. 1992; Nietzel et al. 2001
).
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Acknowledgments |
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The continuous support of the Carl Zeiss GmbH (Jena, Germany) is gratefully acknowledged. Dr. M. Rocchi (Bari, Italy) kindly provided the BAC probe b35B4.
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Footnotes |
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Literature Cited |
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