Page 195 - An Introduction to Microelectromechanical Systems Engineering

174 MEMS Applications in Life Sciences

end of one nucleotide connects to the 5’ end of the next nucleotide. This essentially
gives directionality to the DNA chain.
Two strands of DNA are joined by weak hydrogen bonds to form the well-
known twisted double-helix structure [6]. The attachment occurs between specific
pairs of nucleotides: guanine bonds to cytosine (G–C), and adenine bonds to
thymine (A–T). This important pairing property is known as complementarity.
Color photography makes a simple analogy to understand complementarity: The
three additive primary colors—red, green, and blue—are in their respective order
complementary to the three subtractive colors—cyan, magenta, and yellow. A posi-
tive photographic print and its negative contain the same image information, even
though the colors of the positive (the additive colors) are different from the colors of
the negative (the subtractive colors). The positive and negative in photography are
analogous to the two complementary strands of DNA in a double helix.

PCR
A primary objective of genetic diagnostics is to decipher the sequence of nucleotides
in a DNA fragment after its extraction and purification from a cell nucleus. This task
is difficult due to the miniscule concentration of DNA available from a single cell. As
a solution, scientists resort to a special biochemical process called amplification to
create a large number of identical copies of a single DNA fragment. The most com-
mon amplification method is the polymerase chain reaction (PCR). Invented in the
1980s by Kary Mullis, for which he was awarded the Nobel Prize in Chemistry in
1993, it allows the replication of a single DNA fragment using complementarity.
The basic idea is to physically separate—denature—the two strands of a double
helix and then use each strand as a template to create a complementary replica.
The polymerase chain reaction begins by raising the temperature of the DNA
fragment to 95ºC in order to denature the two strands. Incubation occurs next at
60ºC in a solution mix containing a special enzyme (called DNA polymerase, an
example of which is Taq polymerase), an ample supply of nucleotides (dNTPs), and
two complementary primers. The primers are short chains of nucleotides previously
synthesized to hybridize—or to specifically match up using complementarity—with
a very small segment of the longer DNA fragment and consequently define the start-
ing point for the replication process. The DNA polymerase enzyme catalyzes the
construction of the complementary DNA strand beginning from the position of the
primer and always proceeding in the 5’ → 3’ direction. Replication of a portion of
the single strand is rapid, proceeding at a rate of about 50 bases per second [8]. The
cycle ends with two identical copies of only the sections between (and including) the
primers, in addition to the starting DNA template. Repetition of the cycle increases
n
the number of identical copies with a factor of 2 , where n is the number of cycles;
thus, after 20 cycles, about one million copies have been created. The efficiency
drops after about 20 cycles [9], but 30 to 40 cycles are typically needed to generate
sufficient product for later analysis.

PCR on a Chip
There are several advantages to miniaturizing the PCR process. Smaller chambers
have a greater ratio of surface area to volume. Surface area affects the rate of heat