Molecular Biology: PCR techniques
Molecular Biology:
PCR Techniques
Author: Prof Estelle Venter
Licensed under a Creative Commons Attribution license.
OTHER PCR’S
Reverse transcription PCR
This is reverse transcription of RNA followed by PCR (RT-PCR) of the cDNA (copy DNA). Since the
polymerase enzymes used in PCR can only act on DNA templates, the RNA is first transcribed to
cDNA using a commercially available reverse transcriptase enzyme. In eukaryotes, most mature
messenger RNA (mRNA) molecules are synthesized with a poly(A) tail, which protects the mRNA
molecule from enzymatic degradation in the cytoplasm and aids in transcription termination, export of
the mRNA from the nucleus, and translation. The primer used in the RT-PCR is often oligo(dT), which
will bind to the poly(A) tail. The newly synthesized DNA template can then be amplified using PCR.
The RT step can be performed either in the same tube with the PCR (one-step RT-PCR) or in a
separate one (two-step RT-PCR)
A useful application of RT-PCR is in measuring the relative amounts of mRNA in different tissues or in
the same tissue at different times. The amount of mRNA in a cell is generally taken to be a reflection
of the activity of the parent gene, so quantification of the mRNA enables changes in gene expression
to be monitored. The latest development in this area is the use of real-time PCR where the
amplification is monitored online and in real-time (see section 4.9).
Random amplification of polymorphic DNA
Random amplification of polymorphic DNA (RAPD) is used to generate fingerprints of genomic DNA
(viruses, bacteria, fungi, plants), and relies on the use of a short arbitrarily chosen primer (Theron,
1998). If the primers used in a PCR are too short then a mixture of amplified fragments will be
obtained. Under normal circumstances this is to be avoided but it is a useful technique in
phylogenetics, the area of research concerned with the evolutionary history and lines of descent of
species and other groups of organisms. The banding pattern seen when the products of PCR with
random primers are electrophoresed is a reflection of the overall structure of the DNA molecule used
as the template. If the starting material is total cell DNA then the banding pattern represents the
organization of the cell’s genome. Differences between the genomes of two organisms whether
members of the same or different species can therefore be measured by PCR with random primers.
Two closely related organisms would be expected to yield more similar banding patterns than two
organisms that are more distant in evolutionary terms. As with many phylogenetic techniques, the
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Molecular Biology: PCR techniques
interpretation of RAPD analysis is highly complex and as yet there is no agreement regarding the way
in which the data should be handled.
Multiplex PCR
Several DNA segments can be simultaneously amplified by using multiple pairs of primers. The
primers must be chosen so that they have similar annealing temperatures. A difference of
approximately 10 °C in the annealing temperature of the two sets of primers may lead to poor or no
amplification for one or the other target (Theron, 1998).
Nested PCR
In nested PCR, two PCRs are carried out. The first PCR is as normal, yielding a primary amplicon.
The primary amplicon is used as a template in the secondary PCR which is carried out for 15 to 30
cycles using a second pair of primers that anneal to an internal area of the first amplicon (nested
primers). This method increases the specificity and effectivity of the amplification by minimizing nonspecific annealing of the two sets of primers (Theron, 1998)
Touchdown PCR
This technique entails starting with a high annealing temperature, which is gradually lowered in the
next cycles. As soon as the temperature is low enough, amplification starts with only very specific
base pairing between the primer and the template. If the temperature is then decreased further, nonspecific binding may occur but non-specific products must compete with the already formed correct
product, resulting in optimal discrimination between specific and non-specific binding. By applying
this technique the sensitivity of the PCR is increased.
Hot Start PCR
Hot start protocols (D’Aquila et al., 1991, Erlich et al., 1991, Mullis 1991) are designed to reduce nonspecific amplification during the initial set up stages of the PCR and can be used for PCR systems
that do not work well under standard conditions. Even brief incubations of a PCR mix at temperatures
significantly below the Tm can result in primer dimer and non-specific priming.
The aim is to prevent at least one of the critical components from participating in the reaction until the
temperature in the first cycle rises above the T m of the reactants. For example in smaller assays one
of the components common to all tubes (e.g. Taq DNA polymerase) can be initially withheld and
added only after the temperature rises above 80 °C during the first denaturing step. Alternatively, a
wax bead can be melted over the bulk of the reaction mix in each tube and allowed to solidify, and the
withheld component can be pipetted on top of the wax cap. The beads melt after the initial
denaturation step, allowing all components of the PCR to mix. Alternatively, the activity of the
polymerase can be inhibited by the binding of an antibody or by the presence of covalently bound
inhibitors. These inhibitors dissociate after a high-temperature activation step, allowing the
polymerase to function normally.
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Molecular Biology: PCR techniques
Real-time PCR
Real-time PCR uses a fluorescent signal to monitor the accumulation of PCR products in a PCR
reaction in real time. The technique reduces the time required for PCR amplification and analysis and
is suited to:

monitor amplification online and in real-time

quickly and accurately quantify results by using different chemistries:

o
SYBR Green I, a dye specific for double-stranded DNA, or
o
Sequence-specific hybridization probes
detect mutations or discriminate between homogeneous and heterogeneous genotypes.
Refer to Sub-module 3 for more information on real-time PCR
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