John M. S. Bartlett.pdf - Bio-Nica.info

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efficiency of the reaction. Although it is theoretically possible to amplify from a single
copy of a sequence, if the reaction conditions are not optimized, such amplification
may not reach a threshold for detection within the average 30-cycle PCR. If the object
of the exercise is to determine the presence or absence of a specific sequence, elements
of quantitation must be built into the reaction design to determine the required level of
sensitivity. Where PCR is to be used to test for the presence of a pathogen for instance,
consideration has to be given to the clinically significant levels. If a single organism
is clinically significant, then clearly the test has to be capable of detecting a single
copy. If, however, 1000 organisms are required for clinically significant event, the
assay need not be as sensitive (1).
2.2. Mixed Template Competition
If the template contains more than one species of DNA capable of being amplified by
the primers, those templates that exist in greater initial concentrations or that amplify
more efficiently may outcompete the remaining templates. For instance, PCR is used to
amplify papilloma virus sequences in cervical cytology specimens. Consensus primers
are used to amplify a broad spectrum of viral strains, which can then be characterized
by restriction digest or probe hybridization. If the patient has a mixed infection, it
is likely that the more abundant strain will be amplified preferentially, resulting in
only one strain being represented in the PCR product (2). This is perfectly adequate to
determine which are the predominant strain(s) within a sample. However, if a complete
description of the strains present is required, PCR should be performed to specifically
amplify individual strains
3. Quantitative PCR: General Considerations
3.1. End-Point Analysis
Unfortunately, the cause of quantitative PCR has not promoted by the use of end-point
analysis. This approach simply amplifies multiple templates under the same set of conditions
and examines the amount of product at the end of the run. Because PCR exhibits a
typical exponential amplification of product, complete with lag phase and plateau, it is
easily possible for widely differing starting template concentrations to yield remarkably
similar final product concentrations. This can be a useful technique but only if great
care is taken to establish an appropriate end point. Reactions should be sampled after
a wide range of cycle numbers, to delineate the exponential phase, and the subsequent
plateau. Tests should then be designed to sample during the exponential phase of
amplification, before primers, dNTPs, and or enzyme become limiting. As with all
quantifications, it is greatly enhanced by the inclusion of a suitable internal control.
3.2. Limiting Dilution
PCR quantitation can be achieved using qualitative end points. For the PCR to
proceed, there must me a theoretical minimum of one template per reaction. Thus,
if a dilution series is performed on the template, a dilution can be reached where an
aliquot of sample taken for PCR has a statistical probability of containing no template
molecules. The higher the initial template concentration, the greater the dilution
required needed to reach that point. This approach has been used with great success
to quantify viral titers (3).