Biomedical Engineering Reference
In-Depth Information
sequences. The oligonucleotide probe has a chain length, typically
a 25-mer, or possibly longer (60-mer). Although longer oligonu-
cleotide probes are more expensive, they can provide several ad-
vantages over shorter sequences. For fluorometry readout, the
smaller size of the target site (20 u 20 μm or less) requires a
high-resolution fluorescence scanner with laser excitation.
Besides these two types of DNA arrays, macroarrays and mi-
croelectronic arrays with a variety of formats have also been in-
vestigated. They are summarized in
Table 1
and some of these are
commercially available. Here, only the spot density distinguishes
the macro- and micro-array: the spot size is 300 μm or larger for
the macroarrays. Choice of any one of these DNA arrays would be
dependent on the research applications and budget. The microelec-
tronic arrays can be considered as a new entry in this field and will
be discussed in the following section.
2. GeneExpressionProfiling
Hybridization of DNA, the process whereby complementary nu-
cleic acid sequences pair by forming hydrogen bonds between
complementary bases (e.g., A-T and G-C), is the core principle
behind microarrays. The noncovalent binding between the two
strands becomes stronger with increasing sequence length; as such,
only strongly paired strands remain hybridized even after some
nonspecific bonding sequences are removed by exhaustive wash-
ing. Most labeling for DNA microarray analysis involves the use
of fluorescence, which possesses the particular benefit of allowing
the simultaneous reading of several experimental parameters in
samples, otherwise known as multiplexing.
Figure 3
shows a typical dual-color microarray experiment
with a cDNA-pair prepared from two specimen materials to be
compared, such as a particular type of cancer cells vs. normal cells.
Commonly used fluorescent dyes include Cy3, which shows fluo-
rescence at 570 nm (green-colored emission) and Cy5 with fluo-
rescence at 670 nm (red-colored emission). To convert the mRNA
samples to the labeled cDNA, reverse transcription is carried out in
the presence of the fluorescent-labeled nucleotide precursors.
These cDNA products are then combined to hybridize on a corre-
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