36 Microarrays and DNA-Chips in the Analysis of Genetic Aberrations
475
genome, that determines the resolution and sensitivity of this technique.
Since the genetic complexity of a target spot is, at least theoretically,
directly proportional to the number of probe nucleotides that it can
bind (as long as the target DNA concentration is not rate limiting), increased complexity results in higher signal strength with a concomitant
loss in resolution. However, absolute signal strength is not the only factor
that determines assay sensitivity. In practice, a host of parameters such as
probe complexity and concentration, label type and density, hybridization
time and temperature, and a variety of factors related to detection mechanism and instrumentation are of equal importance (Muller 2001).
For diagnostically relevant CGH applications, the hybridization probe
mixture typically contains on the order of 200 - 400 ng of total human
DNA from each of the test and reference tissue. Thus, the total number
of available probes for each target spot is relatively low, placing much
higher demands on the sensitivity of this system than what is needed
for standard FISH. The GenoSensor™ System (Vysis, Inc.) was the first
commercial system to meet these high demands on sensitivity with the
development of a unique chromium chip surface, small arrays of PAC
or PI clones, and a novel Large Field Multicolor Imaging System (Che
et al. 2001). While the sensitivity of this system was shown to be sufficient
for the reliable detection of monosomic and trisomic regions in clinical
material (Bao et al. 1999; Lese et al. 1999), the low resolution of these large
genomic clones still limits the use of this technology. More recently this
limitation was overcome by the use of cDNA arrays for CGH applications
(Pollack et al. 1999; Monni et al. 2001). We describe here a protocol for
array CGH that provides for improved sensitivity, allowing the use of
cDNA arrays with probe concentrations of less than 80 ng sample
DNA per Jll of hybridization solution.
Outline
As in CGH, the DNA from a sample tissue is compared to normal human
DNA in a dual color hybridization that includes the following major steps:
- Extract DNA from a sample tissue
- Label sample DNA with a fluorescent dye (e.g., Cy3-dCTP) and reference DNA with a different color fluorescent dye (e.g., CyS-dCTP) by
primer extension with DNA polymerase
475
genome, that determines the resolution and sensitivity of this technique.
Since the genetic complexity of a target spot is, at least theoretically,
directly proportional to the number of probe nucleotides that it can
bind (as long as the target DNA concentration is not rate limiting), increased complexity results in higher signal strength with a concomitant
loss in resolution. However, absolute signal strength is not the only factor
that determines assay sensitivity. In practice, a host of parameters such as
probe complexity and concentration, label type and density, hybridization
time and temperature, and a variety of factors related to detection mechanism and instrumentation are of equal importance (Muller 2001).
For diagnostically relevant CGH applications, the hybridization probe
mixture typically contains on the order of 200 - 400 ng of total human
DNA from each of the test and reference tissue. Thus, the total number
of available probes for each target spot is relatively low, placing much
higher demands on the sensitivity of this system than what is needed
for standard FISH. The GenoSensor™ System (Vysis, Inc.) was the first
commercial system to meet these high demands on sensitivity with the
development of a unique chromium chip surface, small arrays of PAC
or PI clones, and a novel Large Field Multicolor Imaging System (Che
et al. 2001). While the sensitivity of this system was shown to be sufficient
for the reliable detection of monosomic and trisomic regions in clinical
material (Bao et al. 1999; Lese et al. 1999), the low resolution of these large
genomic clones still limits the use of this technology. More recently this
limitation was overcome by the use of cDNA arrays for CGH applications
(Pollack et al. 1999; Monni et al. 2001). We describe here a protocol for
array CGH that provides for improved sensitivity, allowing the use of
cDNA arrays with probe concentrations of less than 80 ng sample
DNA per Jll of hybridization solution.
Outline
As in CGH, the DNA from a sample tissue is compared to normal human
DNA in a dual color hybridization that includes the following major steps:
- Extract DNA from a sample tissue
- Label sample DNA with a fluorescent dye (e.g., Cy3-dCTP) and reference DNA with a different color fluorescent dye (e.g., CyS-dCTP) by
primer extension with DNA polymerase