Biology Reference
In-Depth Information
conducting PCR, the number of positive and negative reactions is counted and
this allows the scientist to estimate how many copies of DNA molecule were in
the sample. This technique is more expensive to conduct than quantitative PCR,
although the use of nanofabrication and microfluidics has reduced the amount
of reagents required and, thus, the cost. Several dPCR machines are commer-
cially available and the use of dPCR has been used to quantify the number of
viruses in individual cells ( Tadmor et al. 2011, White et al. 2012 ) or the differ-
ential expression of alleles ( Pekin et al. 2011, Whale et al. 2012 ). dPCR provides
a high resolution; you can distinguish between two and three copies using 200
chambers, but need 8000 chambers to distinguish between 10 and 11 copies.
dPCR does not require the same level of calibration or controls as qPCR, but arti-
facts can occur if too many copies occur in the chambers (i.e., the DNA was not
sufficiently diluted), inhibitors prevent reactions from occurring, and specificity
can be poor if pseudogenes are present.
8.5 Some Research Applications
The PCR can be applied to a diverse array of both basic and applied problems
( Table 8.6 ). Protocols for the different methods are available in topic ( Erlich 1989,
Erlich et al. 1989, Innis et al. 1990, Ausubel et al. 1991, McPherson et al. 1991,
Sambrook and Russell 2001, Gariepy et al. 2007 ) or individual journal papers.
The following examples provide evidence of the versatility of the PCR, but are
only an abbreviated introduction to the diversity of applications to which this tool
can be applied. Modifications of the PCR continue to be made to resolve diagnos-
tic, ecological, evolutionary, genetic, and developmental biology questions.
8.5.1 Amplifying Ancient DNA
The film Jurassic Park implied it was possible to amplify dinosaur DNA from
insects preserved in amber; this captured the imagination of the public and
created a climate in which the PCR was perceived to be an unusually power-
ful key to analyzing the past. Subsequently, the PCR was used to amplify DNA
fragments from a number of insects preserved in ancient amber. Unfortunately,
these results have been controversial ( Box 8.1 ), as have been the results of
amplifying dinosaur DNA ( Austin et al. 1997a,b; Rollo 1998; Hofreiter et al. 2001;
Hebsgaard et al. 2005 ).
The most common ancient DNA amplified by the PCR is usually mitochon-
drial DNA because it is so abundant. However, this abundance makes it easy
to contaminate the ancient sample with modern mitochondrial DNA. The
amplification of ancient DNA remains highly controversial because technical
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