1344

Robert E. Levin

4.1 THE POLYMERASE CHAIN REACTION (PCR)

4.1.1 Introduction
The Polymerase Chain Reaction (PCR) is one of the most powerful analytical techniques
ever developed. It allows segments of minute amounts of double stranded DNA to be
amplified several millionfold in several hours. Its most notable application to foods is for
the detection of low numbers of food borne pathogenic and toxigenic bacteria in a wide
variety of food products in addition to confirming the identification of such organisms
isolated from food.
4.1.2 Requirements for the PCR
The polymerase chain reaction uses repeated temperature cycling involving template denaturation, primer annealing, and the activity of DNA polymerase for extension of the annealed
primers from the 3 ends of both DNA strands (Figure 4.1). This results in exponential amplification of the specific target DNA sequence. The availability of the thermostable Taq DNA
polymerase, from the extreme thermophile Thermus aquaticus greatly facilitates repeated
thermal cycling at ~95C for template denaturation, without having to repeatedly add a less
thermally stable DNA polymerase after each cycle. The notably high optimum temperature
for Taq polymerase activity (7580C) allows high extension temperatures (7275C) which
when coupled with a high annealing temperature (5065C) and denaturation at 95C
increases specificity, yield, and sensitivity of the PCR reaction (1). Polymerase chain reactions are usually performed in 0.5 mL or 0.2 mL thin walled polyethylene PCR tubes containing 50 l total reaction volume. The availability of second generation thermal cyclers with
heated lids has eliminated the previous need for overlaying the reaction volumes with 50 l
of mineral oil to prevent evaporation. The four deoxynucleotide triphophosphates (Table 4.1)
are presently available commercially premixed. Variables that require optimization include
components 5, 6, 7, 8, and 9 in Table 4.1. The concentration of MgCl2 is particularly critical.
Innis and Gelfand (2) have discussed the optimization of PCR in detail. Most thermal cycling
of PCR encompasses 35 cycles; rarely are more than 35 cycles of benefit.
A typical thermal cycling protocol is given in Table 4.2. After an initial denaturation
step at 95C, steps 2, 3, and 4 are then sequentially performed for 35 cycles, followed by
step 5 at 72C to ensure that the final round of strand synthesis at high substrate concentration is completed. The 6th step, involving reduction of the temperature to 4C, is used to
terminate all reactions for convenient holding until agarose gel electrophoresis is performed. The time required to traverse from one temperature to another is referred to as the

A strand
3

Extension

5-primer #2

Figure 4.1

Extension

B strand

Amplification of a known target sequence with a set of two primers.

2006 by Taylor & Francis Group, LLC

3-primer #1