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Qualitative and Quantitative PCR 181
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Qualitative and Quantitative PCR
A Technical Overview
David Stirling
1. Introduction
The nature of the polymerase chain reaction (PCR) process lends itself well to
qualitative determinations. It transforms very small quantities of analyte into the realms
of bucket chemistry, allowing specific gene portions to be directly visualized with
ethidium bromide and ultraviolet light. It was these approaches that were first to be
exploited in DNA analysis. The early PCR tests were for the presence or absence of a
gene, transcript, or by inference whole organism (e.g., pathogen identification). These
qualitative tests rapidly evolved to distinguish between related genes or organisms and
accelerated the whole process of molecular taxonomy.
In contrast to this global utility and acceptance, the use of quantitative PCR has
grown more slowly, for similar reasons. A chain reaction by definition is intuitively out
of control. Surely the vagaries of chaos theory will obliterate any hope of quantitation;
it matters not if the butterfly flaps once or one thousand times. Despite these early
misgivings, a great many successful quantitative PCR protocols have been developed
and gained general acceptance.
2. Qualitative PCR
There a number of considerations that should be taken into account when performing
qualitative PCR. Careful thought at the outset of the design process can avoid a great
deal of subsequent frustration. When the PCR product contains the expected amplicon
(assuming all negative controls have been included and are negative), it is safe to
assume the template was present in the starting sample. However, the converse is not
true. There are a number of common causes for the failure of qualitative PCR other
than the absence of template.
2.1. Template Concentration
Even the most basic qualitative PCR is dependent on quantitative changes in template
concentration. The whole process is based on an ideally exponential amplification
of starting material to a point where it can be readily seen and manipulated. Clearly,
this ability will be influenced by both the starting concentration of template and the
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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