DNA Amplification by the Polymerase Chain Reaction: Molecular Biology Made Accessible - Insect Molecular Genetics - page 335

8.4.12 Quantitative PCR
Methods have been developed to quantify the amount of DNA or RNA pres-
ent in a sample ( Arnheim and Erlich 1992, Siebert and Larrick 1992, Foley et al.
1993, Sambrook and Russell 2001, Bustin 2004, Baker 2011 ). Quantitative PCR
requires some form of standard with which the target sequence concentration
is compared. For example, estimation of the number of amplified esterase genes
in insecticide-resistant mosquitoes used a nonamplified esterase gene as an
internal control ( Weill et al. 2000 ). Quantification of infection of fleas with the
plague bacterium Yersinia pestis was based on standard, curve-based, competi-
tive PCR ( Hinnebusch et al. 1998 ). This quantitative PCR method was found to
be equally accurate and precise as a colony-count method when evaluated using
mock samples and laboratory-infected fleas.
Quantitative PCR is more difficult than other types of PCR due to the nature
of the PCR. Because PCR is an exponential (or nearly so) process, small differ-
ences in efficiency at each cycle can lead to large differences in yield. Anything
that affects exponential amplification can disrupt quantitation. Thus, different
amounts of inhibitors in samples containing the same amount of template DNA
could result in different amounts of product, as could small differences in effi-
ciency between the primer pairs used to amplify the standard (control) and tar-
get sequences.
Quantification of amplified products can be achieved by gel electropho-
resis or by fluorescently labeled primers quantified with an automated DNA
sequencer, fluorometry, analysis of gel images stained with ethidium bromide or
other intercalating dyes, or measurement of radioactivity incorporated during
amplification ( Sambrook and Russell 2001 ). Another method for quantitating
PCR products is through real-time PCR (see Section 8.4.14) or TaqMan PCR (see
Section 8.4.16).
8.4.13 Random Primers
A method similar to AP-PCR was developed when Williams et al. (1990) demon-
strated that genomic DNA from diverse organisms could be amplified using a single
short (9- or 10-nt) primer composed of “random” oligonucleotides ( Figure 8.6 ). The
“random primers” can be designed without the experimenter having any genetic
information for the organism being tested. The only constraints are that the prim-
ers should have 50-80% G + C content and no palindromic sequences. Different
random primers used with the same genomic DNA produce different numbers
and sizes of PCR products ( Ellsworth et al. 1993, Kernodle et al. 1993, Meunier and
Grimont 1993, MacPherson et al. 1993, Williams et al. 1993 ). The amplified DNA can