Gene Cloning

356 Gene Cloning
detected by autoradiography or labeled with biotin and detected by chemiluminescence
(Box 5.3). When using a glass-based array or gene chip the
cDNA is labeled with a fluorescent dye and detected by a specialized
machine, called a micro-array scanner, which uses a laser to scan the slide.
The laser excites the dyes at their respective excitation wavelengths and a
detector measures the emission signal from the excited dyes as the laser
passes each spot.
Figure 11.22 is a schematic representation of a glass-based array experiment.
RNA is isolated from control and test cells, and the RNA is used as
template for cDNA synthesis using random hexanucleotide primers in the
presence of dUTP labeled with one of two fluorescent dyes, Cy3 and Cy5.
Cy3 is used to label the control sample and Cy5 is used to label the test
sample. Random hexanucleotides are a population of oligonucleotides six
bases in length with random sequence; using random hexanucleotides
means that all RNA molecules should be reverse-transcribed into cDNA.
The samples are combined and the mixture of labeled cDNAs is hybridized
to the glass array. The array is then scanned using a micro-array scanner.
Transcript levels in the control and test samples are measured as Cy3 or
Cy5 fluorescence respectively. By comparing the relative Cy3 and Cy5 fluorescence
it is possible to determine if genes are up-regulated, downregulated,
constitutively expressed or not expressed under the two conditions
tested. If the Cy3 signal is represented as green and the Cy5 signal as
red, overlaying the fluorescent scans gives different colored spots. Spots
corresponding to genes expressed in both conditions will be yellow (i.e.
green + red), genes induced or upregulated in the control conditions will
appear as green spots, and genes induced or upregulated in the test conditions
will appear as red spots. Spots that give no signal from either dye after
scanning represent genes that are not transcribed under either condition.
Q11.10. In a micro-array constructed with oligonucleotides, should the
oligonucleotide probes be sense or antisense, i.e. the same sequence as the
RNA or the template strand of the DNA?
Q11.11. Could an oligonucleotide array be used to analyze the labeled
RNA from a nuclear run-on experiment?
Proteomics
Proteomics is a term that has been coined to describe the analysis of all
proteins present in the cell. The proteome is the complete set of proteins
produced by a cell or organism at any one time. The proteome, like the
transcriptome, is very dynamic, since different genes are being transcribed,
translated and subject to different post-translational modification at any
given time in a cell. As you will learn, proteomics techniques cannot pro-