Setting Up a Polymerase Chain Reaction Laboratory - Clinical Applications of PCR - page 11

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Setting Up a Polymerase Chain Reaction Laboratory
Y. M. Dennis Lo and K. C. Allen Chan
Summary
One of the most important attributes of the polymerase chain reaction (PCR) is its
exquisite sensitivity. However, the high sensitivity of PCR also renders it prone to false-
positive results because of, for example, exogenous contamination. Good laboratory
practice and specific anti-contamination strategies are essential to minimize the chance
of contamination. Some of these strategies, for example, physical separation of the areas
for the handling samples and PCR products, may need to be taken into consideration
during the establishment of a laboratory. In this chapter, different strategies for the
detection, avoidance, and elimination of PCR contamination will be discussed.
Key Words: False-positive PCR; anti-contamination strategies.
1. Introduction
One of the most important attributes of the polymerase chain reaction (PCR)
is its exquisite sensitivity. However, this high sensitivity has also given PCR
its main weakness, namely, its tendency to produce false-positive results owing
to exogenous contamination ( 1 , 2 ) . Contamination avoidance is therefore the
single most important consideration when setting up a PCR laboratory ( 3 ) ,
especially one designed to generate diagnostic information ( 4-7 ) . In many situ-
ations, precautions that are normally taken in the handling of microbiological
materials are equally applicable to PCR-related procedures ( 7 ) .
2. Sources of Contamination
There are four main sources of PCR contamination. The most important one
is PCR products from previous amplifications, the so-called carryover con-
tamination ( 3 ) . Because of the enormous amplification power of PCR and its
ability to generate up to 10 12 product molecules in a single reaction, this is the