Biology Reference
In-Depth Information
from binding nonspecifically to the wafer. A carefully designed
opaque mask is placed over the wafer, and a light is turned on,
deprotecting the exposed parts of the wafer. The deprotected spots now
have free hydroxyl groups on their surfaces, whereas the areas under
the mask do not. The first nucleotide in the sequence can now be added,
and it will bind to the free hydroxyl groups. Each nucleotide to be
added will also carry a light-sensitive protecting group. Excess
nucleotide will be removed; a new mask will be applied; and the light
will shine again, deprotecting only those areas not covered by the new
mask. Now the second nucleotide to be added will be able to bind to
the free hydroxyl groups, some of which will be on the wafer, and some
of which will be on the first nucleotide. The process will be repeated
again and again, until each spot on the wafer bears an oligonucleotide
that is 25 nucleotide units long. This process allows extremely high
feature density, as is shown in graphic form in Figure 12-6(A).
Figure 12-6(B) shows a finished GeneChip. W
The technologies for microarray production are constantly evolving.
NimbleGen Systems, Inc., for example, uses a technique called maskless
photolithography, utilizing tiny movable mirrors to direct the light to
specific spots, rather than masks to block it. Oligonucleotides may also
be synthesized in situ through the use of inkjet printers, with the printer
head delivering the required nucleotides in the desired sequence. This is
the method used by Agilent Technologies, Inc.
Regardless of the arraying technology used, the manufacturer must
decide which specific oligonucleotides should be synthesized in each
microarray position. The goal is to produce a microarray that represents
all of the genes of an organism. This process has been greatly facilitated
by the success of the Human Genome Project, and of sequencing
efforts involving the genomes of other species. To make it possible
to determine unambiguously which genes are being expressed,
Affymetrix has designed its GeneChips W to contain multiple
oligonucleotides from each gene, but to avoid those oligonucleotide
sequences that are common to more than one gene. Agilent uses longer
oligonucleotides (60 bases long) to accomplish this goal. Other controls
are also built into microarray methods, so that one may be confident
of the validity of the results. For additional information, see Eisen
and Brown (1999), Causten et al. (2003), and Krawetz and Womble
(2003).
C. Using the Microarray: Hybridization and Scanning
FIGURE 12-6.
Panel A: Graphic representation of a GeneChip W
microarray, showing the high density of features
possible with photolithography. Each tiny square
represents a different oligonucleotide. Panel B:
Affymetrix GeneChip W probe array. Image
courtesy of Affymetrix, Inc.
Before being used, the microarray will require some additional
processing, which may include blocking and denaturation. Blocking will
inactivate any free reactive groups that might bind the labeled cDNAs
nonspecifically, reducing background fluorescence. Microarrays with
Search WWH ::




Custom Search