Polymerase chain reaction - An Introduction to Forensic Genetics - page 56

will become dissociated): for each A or T in the primer 2 ◦ C is added to the
melting temperature and for each C or G 4 ◦ C is added; Cs and Gs will bind to
the complementary nucleotide with three hydrogen bonds and are therefore more
thermodynamically stable. To estimate the annealing temperature 5 ◦ C, is subtracted
from the melting temperature. PCR primers can be designed manually or using more
sophisticated algorithms with the aid of software such as Oligo [26], Primer3 [27]
and OligoCalc [28].

Magnesium chloride, nucleotide triphosphates and reaction buffer

MgCl 2 is a critical component of the PCR. The primers bind to the template DNA to
form a primer - template duplex: MgCl 2 stabilizes the interaction. The concentration
of MgCl 2 is typically between 1.5 mM and 2.5 mM; the template - primer stability
increases with higher concentrations of MgCl 2 . A balance is required between the
concentration of MgCl 2 and the annealing temperature. The Ta q

polymerase also

requires magnesium to be present in order to function.
The building blocks for the PCR are deoxynucleotide triphosphates, which are
incorporated into the nascent DNA strand during replication. The four nucleotides are
in the PCR in equal concentration, normally 200 µ M. The reaction buffer maintains

optimal pH and salt conditions for the reaction.

The PCR process

The PCR amplifies specific regions of template DNA. The power of the technique is
illustrated in Table 5.1. In theory, a single molecule can be amplified 1 billion-fold
by 30 cycles of amplification; in practice, the PCR is not 100% efficient but does
still produce tens of millions of copies of the target sequence [21].
The amplification of DNA occurs in the cycling phase of PCR, which consists of
three stages (Figure 5.2): denaturation, annealing and extension. In the denaturation
stage the reaction is heated to 94 ◦ C; this causes the double-stranded DNA molecule
to 'melt' forming two single-stranded molecules. DNA melts at this temperature
because the hydrogen bonds that hold the two strands of the DNA molecule together
are relatively weak. As the temperature is lowered, typically to between 50 ◦ Cand
65 ◦ C, the oligonucleotide primers anneal to the template. The primers are in molar
excess to the template strands and bind to the complementary sequences before the
template DNA reassociates to form double-stranded DNA. After the primers have
annealed, the temperature is increased to 72 ◦ C, which is in the optimum temperature
range for the Ta q polymerase. Nucleotides are added to the nascent DNA strand at
the rate of approximately 40 - 60 per second [29, 30]. The enzyme catalyses the
addition of nucleotides to the 3 ends of the primers using the original DNA strand
as a template; it has a high processivity, catalysing the addition of approximately 50
nucleotides to the nascent DNA strand before the enzyme dissociates; several Ta q
polymerase enzymes will associate and disassociate during the extension phase of
longer PCR products.