16 Comparative Genomic Hybridization
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blocked, then labeled repeat sequences will contribute fluorescence intensity to the result, and the sensitivity of CGH to changes in copy number of
unique sequences will be reduced. If repeat sequences are not well
blocked, heterochromatic regions will be brightly stained by one or
both tluorochromes. This can often be resolved by changing the lot of
CoT-l DNA or by changing the reference DNA.
If the hybridization signal appears dim, inadequate probe concentration could be the problem. To overcome this, the hybridization can be
repeated using more probe, or the DNA can be relabeled. Dim hybridization can also be caused by inadequate illumination. It is critical to make
certain that the microscope is properly aligned and equipped with proper
excitation and emission filters for the dyes being utilized.
System equipment (12). Copy number can be grossly evaluated by direct Digital imaging
visualization, but image analysis is far more sensitive and specific. Quantitative information to determine DNA copy number can only be obtained
using digital imaging. A number of companies provide systems that collect
and analyze CGH data. The system operator needs to be well versed in
theory and performance: casual use is not recommended. A system generally consists of (1) a high quality fluorescence microscope equipped with
high numerical aperture, plan corrected, chromatically corrected objectives; (2) dual or triple-band dichroic mirrors/emission filter blocks,
usually in a filter slider or wheel, for accurate color registration between
test and reference images. If each position for each color in the test and the
reference images are not equivalent, then the numerator and denominator
of fluorescence ratios may refer to different chromosome positions. This
will produce inaccurate fluorescence ratios. Post processing the image files
can produce accurate registration if conventional single-band filter combinations are utilized; (3) a properly selected CCD camera; (4) computer
hardware and software to control image acquisition, process data, store
data, and display results. The software should provide manual control
over the acquisition time for each fluorochrome, the ability to display individual and color composite images, and the capacity to store raw image
data in a convenient file format. Systems presently available have specialized software that allow automatic image processing and provide easy
manipulation of image data. However, the operator should be familiar
with the software and with expected data since he or she must confirm
chromosome identification and choose appropriate metaphases and chromosomes for analysis.
Image quality (13). The images collected for CGH analysis should have
certain characteristics. These include (1) low signal intensity in the het-
211
blocked, then labeled repeat sequences will contribute fluorescence intensity to the result, and the sensitivity of CGH to changes in copy number of
unique sequences will be reduced. If repeat sequences are not well
blocked, heterochromatic regions will be brightly stained by one or
both tluorochromes. This can often be resolved by changing the lot of
CoT-l DNA or by changing the reference DNA.
If the hybridization signal appears dim, inadequate probe concentration could be the problem. To overcome this, the hybridization can be
repeated using more probe, or the DNA can be relabeled. Dim hybridization can also be caused by inadequate illumination. It is critical to make
certain that the microscope is properly aligned and equipped with proper
excitation and emission filters for the dyes being utilized.
System equipment (12). Copy number can be grossly evaluated by direct Digital imaging
visualization, but image analysis is far more sensitive and specific. Quantitative information to determine DNA copy number can only be obtained
using digital imaging. A number of companies provide systems that collect
and analyze CGH data. The system operator needs to be well versed in
theory and performance: casual use is not recommended. A system generally consists of (1) a high quality fluorescence microscope equipped with
high numerical aperture, plan corrected, chromatically corrected objectives; (2) dual or triple-band dichroic mirrors/emission filter blocks,
usually in a filter slider or wheel, for accurate color registration between
test and reference images. If each position for each color in the test and the
reference images are not equivalent, then the numerator and denominator
of fluorescence ratios may refer to different chromosome positions. This
will produce inaccurate fluorescence ratios. Post processing the image files
can produce accurate registration if conventional single-band filter combinations are utilized; (3) a properly selected CCD camera; (4) computer
hardware and software to control image acquisition, process data, store
data, and display results. The software should provide manual control
over the acquisition time for each fluorochrome, the ability to display individual and color composite images, and the capacity to store raw image
data in a convenient file format. Systems presently available have specialized software that allow automatic image processing and provide easy
manipulation of image data. However, the operator should be familiar
with the software and with expected data since he or she must confirm
chromosome identification and choose appropriate metaphases and chromosomes for analysis.
Image quality (13). The images collected for CGH analysis should have
certain characteristics. These include (1) low signal intensity in the het-