Biomedical Engineering Reference
In-Depth Information
determination of the variations in the gene expressions. DNA microarray tech-
nology evolved from the use of solid substrates in performing Southern blot ex-
periments. The basic principle of operation of a DNA-microarray consists of three
main steps (Figure 9.19):
1.
Development of the microarray chip.
That is, the immobilization of dif-
ferent DNA sequences onto different positions (probes). DNA probes
representing many (hundreds to thousands) genes are positioned by two
approaches:
a. The most common approach consists of an array of contact-printing
needles that are first immersed into a multiwell plate holding the sev-
eral probe solutions and then is positioned onto the chip on an acti-
vated substrate such as a glass slide where the needles leave the probe
spots by contact printing.
b. To improve the signal-to-noise ratio, it is crucial to maximize the label
density and minimize nonspecific binding with stringency wash condi-
tions. To achieve higher probe densities, more sophisticated techniques
are used and in these cases the DNA probes are synthesized directly in
situ (i.e., onto the chip). The two most representative techniques are
based on photolithographic processes, where protection groups are
used to control the growth of the DNA molecules, and removed by
means of UV light.
2.
Hybridization.
After carrying out the biological experiment of interest, in
patients, animals, or in vitro cell culture, the nucleic acid to be analyzed is
isolated. The nucleic acid is then labeled by attaching a fluorescent dye and
brought in contact with the probes by flooding the chip with the sample
solution. Due to the base-pairing ability, nucleic acids hybridize to the gene.
3.
Readout.
After washing away the microarray to remove all nonbound
molecules, the hybridized microarray is scanned to acquire the fl uorescent
images as emission from the label incorporated into the nucleic acid(s) hy-
bridized to the probe array. Generated colored images are then analyzed
through the use of image analysis software. Since the sequence and position
of each probe on the array is known, the identity of the bound target nu-
cleic acid after hybridization reactions is determined. Based on fl uorescence
intensities that are proportional to the number of genes bound in each spot,
the expression levels of particular genes are determined.
Cells or tissues
Harvest genetic material (DNA, RNA)
Gene chip
Readout
Hybridize
to Array
Label
Figure 9.19
A schematic showing the steps in the DNA microarray technology.
