Biochips – fundamentals and applications - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 315

100 100

Excitation Emission Excitation Emission

80 80

Cy3 Cy5

60 60

40 40

20 20

0

0

400 450 500 550

Wavelength (nm)

600 650 700 500 550 600 650

Wavelength (nm)

700 750 800

FIGURE 11.7

Excitation and emission wavelengths of Cy3 and Cy5.

be the only light detected by the detector. As can be seen from Fig. 11.7, the wave-
length difference between the excitation and emission light is about 24 nm.
Due to the small size of the hybridization array and the small amount of target
present, it is a challenge to acquire the signals from a DNA array. These signals must
fi rst be amplifi ed before they can be detected by the imaging devices. Signals can be
boosted by two means; namely, target amplifi cation and signal amplifi cation. In target
amplifi cation, the amount of target is increased to enhance signal strength while in sig-
nal amplifi cation; the amount of signal per unit target is increased.

11.2.4.1 Target amplifi cation
Low gene expression or small sample size results in signal strength that is too weak to
be detected. Target amplifi cation assists in boosting the signal strength by using tech-
niques like polymerase chain reaction and rolling circle amplifi cation technology to
increase the amount of target DNA. This leads to an increase in signal.

Polymerase chain reaction (PCR)
Polymerase chain reaction is a method invented by Kary Mullis [16] for creating mul-
tiple copies of DNA through repeated cycles of denaturing, annealing, and synthesiz-
ing driven by DNA polymerase. Specifi c nucleotide sequences are amplifi ed without
the use of living organisms. PCR is a quick, easy, and effi cient technique for amplify-
ing any particular segments of the DNA. Initially, DNA polymerase was taken from
bacterium E. coli . The doubled stranded DNA is heated at 96ºC in order to separate it
into two single strands. At this temperature, the DNA polymerase is destroyed and new
enzymes have to be added at the start of every new cycle. Replenishing the destroyed
E. coli requires much time and effort, thus rendering PCR through DNA polymerase
taken from bacterium E. coli ineffi cient. Saiki et al. [17] experimented and report that
bacterium T. aquaticus (Taq) is subsequently selected to amplify nucleotide sequences
as the Taq polymerase thrives well at a temperature over 110ºC. Taq polymerase does
not breakdown when it is heated with the double-stranded DNA during denaturing.
PCR takes place in a thermal cycler (as shown in Fig. 11.8) which is able to change