Biomedical Engineering Reference
In-Depth Information
DNA sample: 5 A G G T C T C G 3
AGGA
AGGT
AGGC
AGGG
GGTA
GGTT
GGTC
AGGT
GGTC
GTCT
TCTC
CTCG
AGGTCTCG
GGTG
GTCA
GTCT
GTCC
GTCG
TCTA
TCTT
TCTC
TCTG
CTCA
CTCT
CTCC
CTCG
FIGURE 11.6
Sequence assembly using overlapping oligonucleotide probes (see Plate 2 for color version).
to the reference sequence is retrieved from a stock of all possible probes of a given
length. Hybridization between the probes and the control and patient samples then takes
place. In the event where a mutation is present, there is a mismatch between the probe
and the DNA sequence; hence, the probes do not bind to the test sample. The overlap-
ping of probes results in a low percentage of positively expressed probes at the muta-
tion sites, indicating that the sequence of the test sample differs from the control DNA.
11.2.4 Labeling
Labeling must be done on the target materials so that during hybridization, the degree
of hybridization can be detected by the imaging devices. Labeling is commonly done
by polymerase reaction, in which a fl uorescent or a radioactive label is used. A labeled
cDNA is created by reverse transcription, allowing for the creation of a label copy of
the target to be hybridized onto the array.
Two of the common labeling dyes used are Cyanine3 (Cy3) and Cyanine5 (Cy5).
These fl uorescence dye molecules emit light when stimulated by a laser. Since there
is a wavelength difference between the excited light and the emitted light, the fl uores-
cence detector is able to fi lter out the excitation wavelength. At such conditions, the
emitted light scattered from the slide is separated from the excitation light and it will
Search WWH ::




Custom Search