Biochips – fundamentals and applications - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 316

FIGURE 11.8 Thermal cycler for PCR. Source: http://www.intlmiss.com/

the temperature inside the reaction tubes to the temperature required for the different
parts of the reaction to take place. For polymerase chain reaction to be possible, fi ve
components, namely, a DNA template, two primers, DNA polymerase, nucleotides,
and a buffer, are needed. The region of DNA to be amplifi ed must be identifi ed and
this region will be contained in a DNA template. Primers are short, artifi cial DNA
strands that match exactly to the region of the DNA that is to be amplifi ed. For a start,
two primers are needed because the double strand DNA is separated into two single
strands. The DNA polymerase will generate copies of any fragments of DNA that
are to be amplifi ed in a suitable environment provided by the buffer. Nucleotides are
needed to build the new DNA.
The PCR process consists of about 20-30 cycles. Each cycle consists of three steps,
namely, denaturing, annealing, and synthesizing. In denaturing, the double-stranded
DNA is heated at 95ºC. At this temperature, the hydrogen bonds between the double
helix are broken, resulting in the complete separation of the DNA template and the
primers. After separation, the temperature is lowered. The primers bind to their com-
plementary bases on the single strand DNA. Temperature at this step is approximately
55ºC. Finally, in the synthesizing step, the DNA polymerase starts reading a template
strand from the beginning of the primer and matches it with a complementary nucle-
otide. Each cycle will take about 1-3 min; the time taken for the third step will depend
on the length of the DNA to be amplifi ed. Repeating the three steps will result in more
DNA as every cycle will double the amount of DNA from the previous cycle. A sche-
matic of the PCR and its timeline is shown in Fig. 11.9 and Fig. 11.10 (see Plate 3 for
color version), respectively.

Rolling cycle amplifi cation (RCA)
Rolling circle amplifi cation (RCA) is an alternative method to polymerase chain reac-
tion; it is also a generic amplifi cation technique that can be used in antibody assays.
Using a replication process similar to that used by viruses, RCA allows the recognition,