8/10/2019 Food Biotechnology, Second Edition(1)

http://slidepdf.com/reader/full/food-biotechnology-second-edition1 1/1

B strand 

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 dena- 

turation, 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 ampli- 

fication 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 ~95°C 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 (75 – 80°C) allows high extension temperatures (72 – 75°C) which 

when coupled with a high annealing temperature (50 – 65°C) and denaturation at 95°C 

increases specificity, yield, and sensitivity of the PCR reaction (1). Polymerase chain reac- 

tions are usually performed in 0.5 mL or 0.2 mL thin walled polyethylene PCR tubes contain- 

ing 50 µl 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 µl 

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 95°C, steps 2, 3, and 4 are then sequentially performed for 35 cycles, followed by 

step 5 at 72°C to ensure that the final round of strand synthesis at high substrate concentra- 

tion is completed. The 6th step, involving reduction of the temperature to 4°C, is used to  

terminate all reactions for convenient holding until agarose gel electrophoresis is per-  

formed. The time required to traverse from one temperature to another is referred to as the  

5′ 

5′-primer #2 

A strand 

Extension 

Extension 

3′-primer #1 

3′ 

3′  5′ 

Figure 4.1 

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

006 by Taylor & Francis Group, LLC