Biochips – fundamentals and applications - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 317

Temp/ ° C

1st cycle

2nd cycle N cycles 30th cycle

95

72

55

Time/min

123456

FIGURE 11.9

Timeline for PCR.

amplifi cation, and detection of signals from the array. One of the inventions by Li et al.
[18] is a novel method of amplifying and detecting DNA, making use of variations of
rolling circle amplifi cation to several detection platforms. In their report, Fire et al. [19]
stated that in RCA, single-strand DNA minicircles are used as templates to perform the
rolling nucleic acid synthesis by certain DNA polymerase at a constant temperature.
Later, in 1998, Lizardi et al. found that the amplifi cation of RCA is rapid, technically
simple, and in the presence of a large excess of wild-type DNA, it allows the detec-
tion of mutations. There are two priming approaches for RCA in DNA diagnostics:
the single-primed and double-primed approaches. In the single-primed approach,
DNA polymerase extends a circle-hybridized primer by continuously synthesizing the
DNA minicircle to replicate its sequence. A report by Liu et al. [20] mentioned that the
RCA products usually consist of about 10 2 -10 3 repeats of sequence that are complemen-
tary to the sequence of the DNA minicircles. As suggested in the name, double-primed
RCA operates with two different pairs of primers. One primer is complementary to
the DNA minicircle. The second primer is targeted to the repeated single-strand DNA
sequences of the original RCA product. Through the series event of multiple hybridiza-
tion, extension of primers, and strand displacement, a double-stranded DNA segment is
formed. A study by Thomas et al. [21] found that double-primed RCA gives a greater
degree of amplifi cation as compared to it single-primed RCA counterpart; in a time frame
of one hour, the double-primed RCA is able to yield 10 9 or more copies greater degree of
amplifi cation as compared to it single-primed RCA counterpart; in a time frame of one
hour, the double-primed RCA is able to yield 10 9 or more copies circular sequence.
Since rolling circle amplifi cation takes place at a constant temperature, there is
no need for the target amplifi cation process to take place in a thermal cycler, which
is required to regulate the temperature for different parts of the reaction. The type of
DNA polymerase to be used in RCA is not limited to thermostable enzymes, like the
PCR-based diagnostics. On the other hand, the RCA method requires the environment
to be free of contaminations as the RCA arrays are highly sensitive. Wiltshire [22]