16 Comparative Genomic Hybridization
215
This causes large fluctuations of the fluorescence intensity ratio in response to relatively small changes in normalized fluorescence intensity.
Controls should be included for each hybridization to monitor the
quality of reagents. An important negative control is a normal versus normal hybridization. The result from this hybridization should produce no
relative gains or losses. DNA from a known DNA sample with DNAs of
interest is an excellent positive control.
After data collection, the quality of averaged profiles should be carefully appraised and unreliable data should be rejected. Three important
items to consider are: (1) the coefficient ofvariation in evaluable regions of
the chromosomes should in general be less than 10-15%. Standard deviations larger than this suggest poor hybridization quality. (2) Difficulties in
defining chromosome ends may result in false gains or losses at the telomeres. This problem becomes apparent if the fluorescence ratio is abnormal at many telomeres in one experiment.(3) Copy number aberrations at
the GC rich regions on chromosomes 1p, 19, and 22 must be interpreted
with caution. Copy number aberrations that occur simultaneously at these
locations in a single hybridization are particularly suspicious. If the hybridizations employ unamplified DNA, inverse hybridizations can be used
to confirm copy number aberrations at these sites. These are repeat hybridizations where the labels for reference and test DNAs have been
switched. The resulting ratios should be the inverse of the originals. If
the hybridizations employ DNA amplified by DOP-PCR, inverse hybridizations do not work at the present time. In this case, the experiment needs
to be repeated and the results confirmed. Other methods of confirmation
such as fluorescence in situ hybridization or quantitative PCR should also
be considered.
Before copy number aberrations in a sample can be determined, cutoffs that define the difference between normal and abnormal must be established. One method is to first evaluate DNA isolated from a set of normal tissues for copy number aberrations. These fluctuations can define the
Fig. 3. Average ratio profiles from DNA utilized in Fig. 2. Chromosome number from 1-22,
X, and Yare to the left of each profile. n represents the number of chromosomes that were
analyzed - 8 for each autosome, and 4 for each sex chromosome. The X axis represents
distance along the chromosome. The hatch mark represents the centromere. The Y axis
is the normalized fluorescence ratio. The dashed line represents a ratio of 1 (seen clearly
at Ip and 19q). The dotted lines represent ratios of 1.5 and 0.5. Note the relatively small
standard deviations over most of each chromosome. Large standard deviations are confined
to centromeres and to heterochromatic regions near the centromeres at chromosomes 1,9,
16, and Y. The relative losses at Ip and 19q are evident
215
This causes large fluctuations of the fluorescence intensity ratio in response to relatively small changes in normalized fluorescence intensity.
Controls should be included for each hybridization to monitor the
quality of reagents. An important negative control is a normal versus normal hybridization. The result from this hybridization should produce no
relative gains or losses. DNA from a known DNA sample with DNAs of
interest is an excellent positive control.
After data collection, the quality of averaged profiles should be carefully appraised and unreliable data should be rejected. Three important
items to consider are: (1) the coefficient ofvariation in evaluable regions of
the chromosomes should in general be less than 10-15%. Standard deviations larger than this suggest poor hybridization quality. (2) Difficulties in
defining chromosome ends may result in false gains or losses at the telomeres. This problem becomes apparent if the fluorescence ratio is abnormal at many telomeres in one experiment.(3) Copy number aberrations at
the GC rich regions on chromosomes 1p, 19, and 22 must be interpreted
with caution. Copy number aberrations that occur simultaneously at these
locations in a single hybridization are particularly suspicious. If the hybridizations employ unamplified DNA, inverse hybridizations can be used
to confirm copy number aberrations at these sites. These are repeat hybridizations where the labels for reference and test DNAs have been
switched. The resulting ratios should be the inverse of the originals. If
the hybridizations employ DNA amplified by DOP-PCR, inverse hybridizations do not work at the present time. In this case, the experiment needs
to be repeated and the results confirmed. Other methods of confirmation
such as fluorescence in situ hybridization or quantitative PCR should also
be considered.
Before copy number aberrations in a sample can be determined, cutoffs that define the difference between normal and abnormal must be established. One method is to first evaluate DNA isolated from a set of normal tissues for copy number aberrations. These fluctuations can define the
Fig. 3. Average ratio profiles from DNA utilized in Fig. 2. Chromosome number from 1-22,
X, and Yare to the left of each profile. n represents the number of chromosomes that were
analyzed - 8 for each autosome, and 4 for each sex chromosome. The X axis represents
distance along the chromosome. The hatch mark represents the centromere. The Y axis
is the normalized fluorescence ratio. The dashed line represents a ratio of 1 (seen clearly
at Ip and 19q). The dotted lines represent ratios of 1.5 and 0.5. Note the relatively small
standard deviations over most of each chromosome. Large standard deviations are confined
to centromeres and to heterochromatic regions near the centromeres at chromosomes 1,9,
16, and Y. The relative losses at Ip and 19q are evident