Biology Reference
In-Depth Information
As a result, Nei and Li's coefficient is recommended for computing genetic simi-
larities with RAPD data, particularly if PCR artifacts are present.
Some of the negative aspects of RAPD-PCR can be eliminated by a method
called
S
equencing
W
ith
A
rbitrary
P
rimer
P
airs (SWAPP) (
Burt et al. 1994
). In
this procedure, amplified random bands are purified from the gel, reampli-
fied with the same two primers used in the initial amplification, and repuri-
fied. One of the primers then is added back and annealed to the product and
sequenced. SWAPP allows polymorphisms in populations to be characterized
at the nucleotide level, eliminates non-Mendelian inheritance, and allows
bands to be produced reliably. The technique requires only small amounts
of low-quality DNA and no prior genetic information for the organism.
Lunt
et al. (1999)
developed a method for isolating microsatellite data from RAPD
fragments that reduced the time and expense of traditional microsatellite iso-
lation methods.
8.4.14 Real-Time PCR
Real-time PCR can quantify gene expression and confirm differential expres-
sion of genes (
Dorak 2006, Bustin et al. 2009, Baker 2011
). Real-time PCR uses
commercially available fluorescence-detecting thermocyclers to amplify spe-
cific nucleic-acid sequences and measure their concentration simultaneously
(
Sambrook and Russell 2001
). Target sequences are amplified and quantified in
the same PCR machine.
Internal standards are not required to quantify the amount of DNA or RNA
present in real-time PCR. The ability to quantify the amplified DNA during the
exponential phase of the PCR, when none of the reaction components are in
limited amounts, results in improved precision in quantification of target
sequences. Real-time PCR can measure the initial concentration of target DNA
over a range of 5 or 6 orders of magnitude. At present, the limit of detection
when fluorescent dyes are used is
≈
10-100 copies of template DNA in the start-
ing reaction (
Sambrook and Russell 2001
). The TaqMan method of real-time PCR
is described in Section 8.4.16.
Significant advantages of real-time PCR include its ability to measure DNA
concentrations over a large range, its sensitivity, its ability to process multi-
ple samples simultaneously, and its ability to provide immediate information.
A disadvantage is that the machines are more expensive than traditional PCR
machines. Real-time PCR is especially useful in large commercial laboratories
that process a large number of samples of a similar type.
Bustin et al. (2009)
rec-
ommend guidelines that will improve the reliability of quantitative PCR data
and allow others to reproduce the work.
