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

198 McDonagh
the dilution method is a semiquantitative system. Another consideration is that several
reactions have to be run if Poisson distribution analysis is to be conducted using the
limiting dilution method. Perhaps the most important consideration is that tube-to-tube
variation is not controlled using this method (1).
1.2. External Standard Curves
This is based on the generation of an external standard curve where a dilution series
of known amounts of template are carried out alongside each run.
1.3. Coamplification of an Internal Reference Template
The problem of tube-to-tube variation can be overcome by the coamplification of
a single-copy cellular gene and the target sequence. This means that both templates
should be affected by any variations of amplification efficiency. However, because
different templates and reactions have different efficiencies, the relative amounts of
product could vary. An added problem is that this system cannot be used to quantitate
extracellular organisms and therefore is of no value in measuring viruses, including
HIV and HCV, in plasma samples (1). As a result, this method will not be considered
further.
1.4. Competitive PCR Using an Internal Control
The use of a competitive internal control that is similar to the target apart from
length or the existence of a restriction site may alleviate the problems encountered with
other methods of qPCR, such as co-amplification of an internal reference template (9).
It will also act as a control for inhibition (10). Both the target and control DNA should
amplify with the same efficiency because they compete equally under given conditions
and the ratio of products will therefore remain constant throughout amplification (11).
The point at which target and control DNA are equal can then be found by direct
comparison using gel electrophoresis and densitometry or by enzyme immunoassay
(EIA). This method has become the method of choice for quantitative assays, but it is
expensive and time consuming because it requires several reactions per sample.
To conduct a competitive PCR, it is necessary to create a template that is virtually
the same as the product to be amplified but which can be identified during the detection
stage. Primer-directed mutagenesis can be used to create a deletion, insert, or substitution
at any point in the fragment (12,13). An outline of this procedure can be seen
in Fig. 1. Two separate reactions are conducted, each using an external primer plus
an internal primer containing the appropriate changes, for example, to create a novel
restriction enzyme (RE) site. This can then be used as a competitive template, which
can be differentiated from amplified wild-type product. The products of these reactions
are purified, denatured, and renatured together then added to a PCR containing the
outer primers. The resulting amplicon contains the mutated site and can be used directly
or cloned to create as much template as required.
The analysis of products is also important. Agarose gel electrophoresis, sometimes
with densitometry, is easy to perform and is suitable for differentiating wild type from
mutated products after digestion with the appropriate enzyme.