Definitive PCR

The Polymerase Chain Reaction
Chapter 1
Introduction
We live in an age where hype and exaggeration have become so pervasive that it is difficult to find
adequate terms for something really extraordinary. Furthermore, impatience, haste and short attention
spans seem to be defining adages for our times, inviting acclaim for technological bandwagons that
briefly promise the earth, but then fail to deliver because the technologies were either conceived in
haste without proper regard for technical and biological concerns or become superseded by the next
technological revolution.
The polymerase chain reaction (PCR) is the antithesis of such technologies and richly merits all the
amplification it attracts. Its conceptual clarity, practical minimalism and ubiquitous applicability
make it the wonder technology of the molecular biology age.
Success has many fathers, and the PCR is certainly no orphan. The truth behind what steps, whose
contributions and which timelines were critical to the invention of the PCR will probably never be
known , but the public face of PCR is Kary Mullis, who was jointly awarded the 1993 Nobel Prize in
Chemistry "for contributions to the developments of methods within DNA-based chemistry" and
specifically for "his invention of the polymerase chain reaction (PCR) method". The other half went
to Michael Smith for his fundamental contributions to site-directed mutagenesis. Whether the rather
romantic story of its invention in Mullis book is how it really happened will forever remain a moot
point. Its influence on modern science, however, cannot be overemphasised and its hold on the
imagination is also worth recalling. Hence it is amusing, and probably telling, that the official Web
Site of the Nobel Prize chooses to highlight amongst the uses of the PCR method a science fiction
application of PCR: its role in the film "Jurassic Park", where it is used to recreate extinct dinosaurs.
It is also funny, and really quite telling, that the site refers to them as giant reptiles.
If PCR initiated a revolution, real-time PCR (qPCR) not only cemented its achievements but extended
them into areas inaccessible to conventional PCR. Its inventor, Russ Higuchi, deserves his place as
an all-time giant of science for realising not only that a fortuitous finding could have such tremendous
implications, but developing all the concepts and practices that are still being followed today.
PCR ingredients
The PCR is a model enzymatic reaction that results in the synthesis of virtually unlimited copies of a
specific DNA from a mixture containing numerous different DNA molecules. A PCR reaction has the
following requirements:
Knowledge of at least some of the sequence of the target DNA molecule. This does not
have to be the exact sequence, and it is possible to amplify sequences that are somewhat dissimilar.
A DNA template. This can be fairly crude and DNA can be quite degraded and very dilute.
Short oligodeoxyribonucleotides known as primers. These are essential, because DNAdependent
DNA polymerases can extend only pre-existing chains; they cannot direct de novo synthesis by
joining two deoxyribonucleoside-5'-phosphates together to make the initial phosphodiester bond. In
general, PCR primers are 18-24 nucleotides in length and are specific to complementary sequences
on opposite strands of their target DNA. However, they can be longer, for example if they have a T7
polymerase promoter site at their 5-ends and may have some mismatches (degenerate primers), as
long as these are not at the 3-end of the primer sequence.
A reaction mixture containing K
+
and Mg
2+
or Mn
2+
, all four deoxynucleoside triphosphates
(dNTPs) and a DNA-dependent DNA polymerase. dUTP is sometimes added to prevent carry-over
contamination
[1]
and the polymerase should be heat stable, although of course the initial experiments
were carried out with a thermolabile enzyme.
A thermal cycler that controls and rapidly varies the temperatures of the PCR reaction mixtures.
Thermal cyclers are now programmable, but the process was initially carried out by manual transfer
of reaction tubes from one water bath or heating block to another. Cycling involves a denaturation
step between 92C and 95C, which breaks the hydrogen bonds holding double stranded DNA
(dsDNA) together, an annealing/polymerisation step usually between 50C and 65C, which allows
optimal hybridisation of primers to their complementary target sequences on the DNA template as
well as their initial or complete extension by the polymerase and an (optional for SYBR Green I)
polymerisation step usually between 70C and 72C, which allows the DNA polymerase to initiate
and extend efficiently towards the primer on the DNAs opposite strand.
Some means of analysing the PCR results; this can be by gel electrophoresis as in legacy, endpoint
PCR or by real-time detection of fluorescent signals that are directly proportional to the number of
amplification products, usually known as amplicons, generated during each amplification cycle.
PCR methodology
At first sight the PCR reaction is rather straightforward:
It is initiated by combining a DNA sample at low concentration with a forward (sense) and reverse
(antisense) primer pair in a 10mM Tris-HCl, pH 8.3, 50mM KCl reaction buffer containing
equimolar ratios of four deoxynucleoside triphosphates, together with Mg
2+
and a thermostable DNA-
dependent polymerase, usually Taq polymerase
Some home-brew buffers contain in addition bovine serum albumin (BSA), - mercaptoethanol
and NaCl, and modified bases such as biotin-11-dUTP and 7-deaza-dGTP may also be included,
depending on the aim of amplification.
This mixture is heated to around 95C for a period of time, which used to be 15 seconds but these
days can be as short as one second for amplification targets up to 500 base pairs (bp). This
denaturation step separates the complementary DNA strands, leaving them single stranded and open
to targeting by the primers that are present in vast excess. The trick here is to heat the sample to the
lowest denaturation temperature for the shortest possible time, so maximising both strand separation
as well as enzyme stability.
The mixture is rapidly cooled to the annealing temperature, which is usually somewhere between
50C and 60C and is held for increasingly short times, with one second perfectly feasible these days
with fast reagents and SYBR Green-based chemistry. If the primers find their complementary
sequences, they base pair with the template DNA, forming a short, double-stranded region.
Primers must possess a free 3'-OH end to which an incoming deoxynucleoside monophosphate is
added by the Taq polymerase. The deoxynucleoside monophosphate to be incorporated is chosen
through its geometric fit with the template base to form a WatsonCrick base pair. As Taq polymerase
catalyses the successive addition of deoxynucleotide units to the 3'-end of the primer, primer and
template complex are stabilised. Many protocols, especially those using probe-based chemistries, use
only two temperatures, as the polymerase has sufficient activity at 60C (approximately 50%) to
complete the polymerisation process and it helps ensure that the probe remains hybridised to its
template until displaced and cleaved by the polymerase.
In the three-step protocol used for conventional and DNA-binding dye-based chemistries, the
temperature is raised to 72C, close to the optimal temperature for Taq polymerase allowing the
polymerase to generate specific amplification products.
In vivo DNA synthesis is always in the 5'3' direction; hence the PCR reaction proceeds to
synthesise a polynucleotide sequence that runs antiparallel and complementary to the template until
each newly synthesised strand reaches the end of the complementary sequence delineated by the 5-
end of the opposite primer.
In theory, this results in a doubling of the amount of original template DNA present in the PCR
solution. Since the product of one cycle serves as the template for the next cycle, PCR leads to the
exponential amplification of the initial DNA template, producing over 1x10
6
copies of a homogenous
PCR product in 20 cycles
[2]
.
Hence, given a known DNA sequence, it is possible to amplify it specifically from every other DNA
molecule that surrounds it.
If the aim is to target RNA, this is achieved by adding a preceding reverse transcription step and
performing the PCR reaction on the resulting cDNA sample. Whilst this allows the detection of
cellular RNAs, including their localisation using in situ RT-PCR, it also opens up the field of
diagnostics to permit the sensitive and specific detection of RNA viruses.
RT-qPCR will be discussed in a separate volume in this series, but it is worth mentioning that the
addition of the reverse transcription step changes the nature of the qPCR assay. It requires careful
quality control of the RNA templates being investigated, assessment for RT-inhibition, and the
variability of the RT step can introduce significant errors and uncertainty into the quantification
cycles (Cqs) recorded at the end of the PCR step. These technical difficulties are exacerbated by
variable analysis methodologies, inappropriate normalisation procedures and non-transparent
reporting in the peer-reviewed literature.
A PCR reaction has three distinct phases:
The early cycles: These require optimal primer specificity. Approximately 10
14
primer molecules
search for their complementary sequences by binding transiently to random sequences, rapidly
dissociating if they are non-complementary and reannealing elsewhere. Specificity is determined by
the annealing conditions, i.e. the temperature and divalent cation concentration, that must be optimised
to favour the hybridisation of perfectly matched duplexes for a period sufficiently long for the
polymerase to form a ternary complex and initiate DNA synthesis from the primer.
The mid cycles: These require optimal amplification efficiency. Here the increasing number of
complementary targets results in more efficient primer scanning, thus allowing the amplification
process operating at maximum efficiency. Ideally, this will result in a doubling of the number of target
sequences during each cycle, although in practice this is confined to a very few cycles.
The late cycles: also known as the plateau phase, must be delayed for as long as possible. This
is when amplification becomes suboptimal due to inhibition of the DNA polymerase, present at
around 3x 10
10
copies, by accumulated target DNA or if not every amplicon is used as a template
because there are more amplicons than polymerase. In addition, at high amplicon concentration the
complementary strands are more likely to find each other and start annealing at a higher temperature
than the primer/template combinations and so will be removed from participation in the next cycle of
the PCR reaction.
Efficiency of amplification
The PCR process is termed a chain reaction because the products from one cycle of amplification
serve as the substrates for the next one. This results in a series of amplicon doubling events with each
cycle of the PCR reaction, defined as 2
n
, where n equals the number of cycles. Theoretically, the
exponential increase in the amount of amplification product is described in equation 1 and plotted in
Figure 3A.
N
a
=N
0
2
n
(1)
N
a
=number of amplicons, N
0
is the initial number of molecules, n is the number of amplification
cycles
This equation denotes the linear relationship between the number of amplified target molecules and
the initial number of target molecules, as shown in Figure 3B.
However, the theoretical efficiency of amplification is not the same as the empirical efficiency and is
rarely 100%. Hence it is necessary to modify equation (1) to add an efficiency correction factor as
shown in equation (2)
N
a
=N
0
(1+E)
n
(2)
E=amplification efficiency.
The exponential nature of the PCR process means that a small change in amplification efficiency can
result in significant differences in the amount of product generated, regardless of whether the number
of initial target molecules was the same. For example, if reactions A and B have amplification
efficiencies of 85% and 95%, respectively, after 40 cycles reaction A would generate a 4.86x10
10
-
fold increase in the amount of target molecules, whereas reaction B would generate a much greater
3.99x10
11
-fold increase, which is more than eight-fold more.
The amplification efficiency is affected by several experimental factors, with primer structure,
amplicon structure, sequence and length as well as sample purity being critical parameters. These
contribute to the observation that the yield of amplification product can differ even if the same target
sequence, cycling conditions and reagents are used
[3-5]
. Moreover, this variability tends to be
unpredictable and can be significant
[6]
.
Importantly, the amount of PCR product levels off as the rate of amplification slows, resulting in the
plateau effect described earlier. The number of cycles required to reach the plateau phase varies and
largely depends on the number of original target molecules, but may also be sequence-dependent.
This variability obscures the linear relationship between initial and final template copy numbers and
so makes conventional PCR unreliable as a quantitative technique.
PCR-theory to practice
The principle of PCR was first described in 1971
[7]
(with an incorrect apostrophe in its title) and it is
worth reading the visionary description of this reaction. There can be no doubt that this paper
describes the essence of the PCR and publicly, albeit theoretically, describes this technique.
So, why did Kleppe et al not pursue their revolutionary and simple concept? Why was the first
practical demonstration of the PCR not published until 1985
[8]
, and then by a different group? Why
did it take another 14 years for this vision to be translated into reality?
Remember that molecular biology was in its infancy, with the first restriction enzyme ( HindII)
coincidentally isolated in 1970. So, what was lacking at that time were various crucial components
we nowadays take for granted, but which at the time of Kleppes thinking about his theoretical
experiment were simply not available. Amongst these there are three elements in particular that stand
out:
1. A reliable, fast and cheap way of preparing oligonucleotide primers of 15 to 25 nucleotides. I
remember watching a post-doc struggling to synthesise an 8-mer back in 1983, with messy chemicals,
manual operations and profusions of bad language.
2. A DNA polymerase that could survive the repeated rounds of heating and cooling without requiring
replenishment after each cycle. Opening the caps of 50 tubes every few minutes to add fresh enzyme
is not really a recipe for reproducible results.
3. Automated thermal cyclers with reliable temperature ramping and holding. No technique, no matter
how powerful, could thrive if it meant sitting for two hours next to a set of waterbaths and transferring
racks of tubes every minute from one to the next.
Oligonucleotide primers
Advances in oligonucleotide synthesis chemistries, coupled to improved purification and quality
control processes, have resolved the first challenge. Together, they have resulted in substantial
increases in primer quality, yield and length, crucially combined with the all-important crash in cost.
Oligonucleotides have become a commodity, purchased in bulk at rock-bottom prices and available
from numerous competing oligonucleotide manufacturers.
The major advance was the substitution of the chloride leaving group present on a phosphite-triester
with the amine leaving group on a phosphoramidite monomer
[9-11]
(also see chapter 1, section 2).
Resulting nucleoside phosphoramidites are stable nucleic acid monomers with an acid-labile
dimethoxytrityl leaving group at their 5-end and a base-labile -cyanoethyl protected 3'-phosphite
group at its 3-end. This modification made it possible to synthesise phosphoramidites in advance,
isolate them as stable solids and store them until required, thus enabling commercial synthesis and
distribution of DNA synthesis reagents.
The first monomer is attached through its 3 carbon to a glass or polystyrene bead with surface holes
and channels. Hence synthesis begins with the 3-most nucleotide and proceeds through a series of
deprotection, coupling, capping, and stabilisation cycles that result in sequential additions to the 5
end of the growing oligonucleotide until the 5-most nucleotide is attached. The high coupling
efficiency (typically >99%) permits the manufacture of long oligonucleotides in excess of 100 bases.
Solid phase synthesis allows excess reagents to be washed away and avoids polymerisation that
would occur in a solution phase reaction. The introduction of tetrazole catalysis
[12]
for
phosphoramidite activation just prior to coupling completed the breakthrough that was essential for
PCR to become a ubiquitous technology, rather than a plaything for chemists.
Thermostable DNA polymerase
The second constraint was removed by the discovery of todays most commonly used DNA
polymerase, identified from Thermus aquaticus, hence its name Taq polymerase, a bacterium that
lives in thermal hot springs and and depends on enzymes that are resilient to inactivation by high
temperatures
[13, 14]
. Taq polymerases half-life is 130 minutes at 92.5C, 40 minutes at 95C and nine
minutes at 97.5C
[14]
. Based on sequence similarity to Escherichia coli DNA polymerase I, Taq
polymerase has been assigned to the A family of DNA polymerases
[15]
and, like E. coli DNA Pol I, it
possesses an intrinsic 53 exonuclease (nick translation) activity
[16]
. Its structure-dependent
single-stranded endonuclease activity allows Taq polymerase to cleave 5 terminal nucleotides of
double-stranded DNA, releasing mono- and oligonucleotides. The preferred substrate for cleavage is
displaced single-stranded DNA, which assumes a fork-like structure; hydrolysis occurs at the
phosphodiester bond joining the displaced single-stranded region with the base-paired portion of the
strand. Taq polymerase has no 35 exonuclease (proofreading) activity
[14, 17]
. Its introduction to
PCR had several important consequences:
PCR could be automated, since there was no longer any need to add DNA polymerase after each
denaturation step.
Tubes no longer needed to be opened after the completion of every PCR cycle. This reduced the risk
of contamination by PCR amplification products, although of course the tubes still needed to be
opened after the PCR reaction was completed. The resulting aerosols certainly contributed to low-
level contamination that was present in every PCR laboratory.
Since Taq polymerase has an optimum reactivity (Vmax) between 70C and 80C and significant
residual activity between 55C and 70C, the polymerisation step can be performed at 72C, rather
than at the original 37C. This results in reduced secondary structures, speedy stabilisation and
extension of annealed primers concomitant with a huge improvement in replication specificity. This
made recourse to a Southern blot less obligatory, since it allowed the viewing of (more or less)
single bands on ethidium bromide stained agarose gels.
The increased processivity of Taq polymerase, 50-80 nucleotides/second at 60C before
dissociating from the DNA template
[18]
, compared with the 20-40 nucleotides/second of Klenow
resultes in the amplification of significantly longer fragments (4,000 bp up to 10,000 bp) than was
possible with Klenow (400 bp).
The absence of a 3-5 (proofreading) exonuclease activity makes Taq polymerase faster than
Klenow.
Taq polymerase generates significantly higher yields of PCR amplicon than Klenow
[19]
.
Taq polymerase is less sensitive to inhibition than Klenow, making for more robust PCR protocols,
although there is some suggestion that it is sensitive to proteolytic degradation.
Thermal cyclers
Thermal cyclers are programmable cycling incubators that automatically and precisely regulate and
change temperatures for DNA denaturation, primer annealing, and primer elongation at defined
intervals. They usually incorporate a thermal block that holds individual tubes, strips of tubes or
microtitre plates. Rapid heat transfer from the heating block to the in-tube sample liquid ensures a
high efficiency of amplification and a thermal processor must enable temperature uniformity for all
samples within an individual run as well as run-to-run repeatability. The inadequacy of early heating
blocks led to the development of water bath as well as rotor-based thermal cyclers and
nanotechnology is beginning to have an impact on the latest thermal cycler designs that incorporate
microfluidic chips with pico- or nanolitre volumes.
Cetus Instrument Systems developed the first thermal cycler, an aluminium block that could be heated
and cooled as required and in a joint venture with PerkinElmer introduced the first fully automated
PCR unit in the 1980s (Mr. Cycle). Why Mr. Cycle? It is not obvious to a nonAmerican, but Russ
Higuchi informed me that it was named after a coffee machine popular in the USA, although its name
was unfortunate if it ever missed a cycle. Another early instrument was a prototype called Baby
Blue (because of its beautiful blue colour) and was devised in 1986 to study HIV. It was the first
model that combined the software controlling the process with the heating and cooling block in one
machine. It is on show at the Science Museum in London and more information is available online.
Successful amplification of a DNA target depends on several variables:
Actual temperatures inside the sample
Uniformity of heating and cooling across the block
Ramp times of the thermal cycler
Degree of convection in the sample
Design of the plasticware holding the samples
Early designs had significant issues with temperature homogeneity
[20]
and accuracy
[21]
, variable
performance
[22]
; even more recent designs do not always perform within the manufacturers
specification
[23]
and performance continues to be limited by spatial variation across the block
[24]
.
Nevertheless, in general todays instruments are not just robust, accurate and capable of very high
throughput, but are also (relatively) inexpensive and easy to use.
Different sample volumes may require an adjustment to the incubation times to maximise thermal
equilibration of the reagents. One of the banes of PCR-of-old was the need to use oil to seal in the
reaction to keep it from condensing inside the lid of the tube. This removes water from the reaction
mixture and so concentrates the salts and other reaction components. Luckily, modern thermal cyclers
have heated lids, which maintain a constant temperature of around 105C. This ensures that the
temperature of the exposed portions of the tubes or wells is raised and keeps condensation to a
minimum. Ramp time, which refers to the time it takes the heated block to change from one
temperature to another, is usually longer than either denaturation or primer annealing times and the
shorter it is the better.
Real-time PCR
How did Russ Higuchi invent qPCR? As he himself recalls, he had been working on a project that
involved the use of biotinylated oligonucleotide primers and streptavidin to determine whether PCR
could be used to generate long branched chains of amplicons. Unfortunately all that appeared to
happen was that DNA precipitates formed, which were visualised by the manual addition of EtBr
after the PCR step, followed by UV illumination. At one stage Russs technician, Bob Griffith,
became fed up with having to add EtBr each time after the PCR and added it to the mastermix
beforehand. On one occasion, whilst looking at a band on a gel, he mentioned to Russ that that
particular PCR reaction had proceeded with EtBr in the reaction, something that should have not been
possible since EtBr is a known inhibitor of DNA polymerases. They immediately did the experiment
again, this time with a no template control (NTC), which did not show any sign of specific
amplification. When they repeated the PCR without the streptavidin and illuminated the reaction tubes
with a UV light, the tube containing target DNA lit up brightly, whereas the NTC did not. Of course
they got very excited, since this suggested that, given the right conditions, addition of EtBr to the PCR
reaction might allow the detection of the amplified DNA through increased fluorescence without the
need to open the reaction tube. Furthermore, it immediately occurred to Russ that a continuous
monitoring of the PCR reaction, rather than endpoint detection, would be a useful feature of this new
methodology.
So he hooked up a thermal cycler to a spectrofluorometer and found that he could indeed follow the
PCR reaction in real time. The output was very simple, with the fluorescence trace at 600nm
recording peaks and troughs corresponding to readings at 50C and 94C, respectively. In addition,
there was a net increase in fluorescence at the lower temperature after each cycle, corresponding to
the increasing amount of amplicon made during each polymerisation step. Together with Bob Watson,
Russ placed a charge-coupled device (CCD) camera so that it looked directly down at the reaction
tubes sitting in a thermal block in a darkroom and illuminated the setup with a UV light. An image
was taken at every cycle of the PCR reaction and the resulting pixel values were combined for each
individual tube. This resulted in the now familiar amplification plots, but because of the different
baseline fluorescence values could not be used for quantification. This is where Russs second
brainwave came in. He realised that if one made the reasonable assumption that baseline fluorescence
was the same for all samples, one could normalise PCR results relative to each other based on their
early cycle fluorescence readings. This generated perfectly overlapping curves, with variation
between Cqs being fewer than 0.2 Cqs.
The third brain wave was the realisation that all PCR reactions have fixed start and stop cycles,
allowing amplification products to catch up with each other. Russ realised that this could be the basis
of a quantitative assay, with perfect quantification conditions right up to the plateau phase. He
devised the concept of a fluorescence threshold value and demonstrated that the number of cycles it
took to cross this threshold is inversely and linearly related to the logarithm of the initial number of
target molecules. These cycle numbers are now known as quantification cycles (Cq) and can be
interpolated to fractions of cycles. If Cqs are compared with the Cqs obtained from a standard curve
with known initial target copy numbers or amounts, the starting target number in each unknown can
be inferred. He also realised that the slope of the standard curve was related to the per-cycle
efficiency of PCR replication. Since 100% efficiency equates to a perfect doubling per cycle, a
twofold dilution of starting template would result in a one-cycle difference between Cqs. If the
efficiency were less than 100%, the difference would be more than 1 Cq. Consequently, the slope of
the calibration curve describes the number of cycles required to make up for the dilution. He
described per-cycle efficiency as:
(10
-1/slope
-1)x100%
This calculation showing the relationship between slope and PCR efficiency for 10-fold dilutions is
still in use today.
Present day chemistries
Present-day qPCR assays utilise three general approaches:
Non-specific DNA-binding dyes that are a further development of the first EtBr-based qPCR
Non-destructive hybridisation-based assays
Combined hybridisation/hydrolysis-based assays
Regardless of chemistry, the amount of fluorescence emitted is directly proportional to the number of
PCR amplicons being synthesised, although the kinetics of fluorescent reporter increases depends on
the type of chemistry used. Increases (or decreases) can be either cumulative, as with hydrolysis
reporters or non-cumulative, as with DNA-binding dyes or hybridisation reporters
[25]
.
DNA-binding dyes
The simplest, cheapest and most widely used approach makes use of the affinity of certain fluorescent
dyes, for example SYBR Green I, for double-stranded (ds) DNA. When in solution and unbound to
DNA, their fluorescence emission is very low when subjected to light of an appropriate wavelength.
The accumulation of amplicon during each PCR cycle results in the binding of increasing numbers of
dye molecules to ds DNA. This induces a conformational change that leads to hugely increased
fluorescence of the excited dye. Hence the effect is like turning up a dimmer switch, where with each
twist of the knob there is a little more light (Figure 1A). As with conventional PCR, the specificity of
the reaction is determined entirely by the primers; hence the principal disadvantage of using this
method is that both specific and non-specific products generate fluorescent signals. Its main
advantages are that there is no need for additional, expensive oligonucleotide probes and that it is
possible to carry out post-PCR melt point analyses to check for the presence of non-specific reaction
products.
Non-destructive hybridisation-based assays
Although non-destructive hybridisation assays include a wide range of different chemistries, in
practice only two or three really matter.
Fluorescently labelled primers become incorporated into ds amplicons and fluorescence is either
increased or decreased. These primers can be simply labelled with a fluorochrome (Figure 1B),
make use of specific pairing by a synthetic bases (Figure 1C), or can be more complex and include a
probe component at their 5-end, which in a unimolecular reaction binds to and reports the presence
of a specific amplicons (Figure 1D).
There are several variants on the theme of a single fluorochrome/single probe that is complementary
to specific targets. Following hybridisation to newly synthesised amplicons, dequenching of the
reporter results in the emission of fluorescence, which is detected either directly (Figure 1E) or is
transferred to a DNA-binding dye via resonance energy transfer and detected as emission at its longer
wavelength (Figure 1F).
A single oligonucleotide probe with donor and acceptor moieties attached to its 5- and 3ends.
Single oligonucleotides behaves like random coils in solution, the ends will come together from time-
to-time, resulting in quenching by fluorescence energy transfer. Binding of the probe to its target locks
it into a linear conformation, prevents interaction between donor and acceptor and results in
fluorescence emission (Figure 1G).
The hybridisation of a pair of non-complementary oligonucleotide probes labelled at their
respective 5 and 3 ends to adjacent sites on a target strand brings a donor and acceptor moiety close
to each other, resulting in resonance energy transfer and detection of fluorescence emission from the
acceptor moiety at a different wavelength
[11]
. (Figure 1H).
Two complementary oligonucleotides, one of which contains a donor fluorochrome at its 5-end, the
other contains an acceptor moiety at its 3end (chapter 3 section 3), are annealed and emit no
fluorescence in the absence of complementary targets. Since small complementary oligonucleotides
displace and bind to each other in a dynamic equilibrium, PCR amplicons compete for binding to the
probes during the PCR reaction. This separates the labelled oligonucleotides and results in
fluorescence emission (Figure 7K).
The probe sequence can contain additional complementary sequences at either end that anneal in the
absence of target to generate a hairpin stem and so bring the terminal fluorochrome/quencher moieties
into close contact. In the presence of target complementary to the probe sequence, a relatively rigid
probetarget hybrid is created, which disrupts the stem structure and separates donor from acceptor,
resulting in fluorescence (Figure 1L).
Combined hybridisation/hydrolysis-based assays
Hybridisation/hydrolysis-based chemistries are variations on the basic theme of attaching fluorescent
donor and acceptor moieties to the same oligonucleotide, which can be either a targetspecific or a
universal probe. These take on a random coil conformation in solution and fluorescence is quenched.
In the most popular embodiment, presence of target results in the dual-labelled probe binding to the
amplicon, followed by cleavage by the 53 activity of Taq DNA polymerase. This results in the
separation of fluorochrome and quencher with concomitant emission of fluorescence (Figure 1M).
A second hydrolysis-based approach uses a primer with an antisense DNAzyme binding/ cleavage
site at its 5-end that becomes activated during the PCR reaction and cleaves a fluorescently labelled
universal probe (Figure 1N).
Alternatively, the target-specific probe may be separated into two sections that together specify a
DNAzyme recognition and cleavage site, so that the universal probe is hydrolysed only in the
presence of a complementary target that is bound by both sections that binds to the universal probe
and cleaves it (Figure 1O).
Within each class, there are numerous variants, most of which will be described in more detail in the
relevant chapters of this book. They provide an immense flexibility for assay design, allowing a wide
choice of chemistries to suit each task on hand.
Figure 1. (overleaf) A. DNA dye-based chemistry. The transformation from free dye in solution (blue) to dsDNA binding (glowing
green) changes the conformation of the dye and results in fluorescence emission (SYBR Green I). B. The hairpin structure of the free
primer with the fluorochrome attached towards the 3-end of the molecule (green) opens up during the PCR, dequenching the
fluorochrome (glowing green) (Lux). C. The 5-end of the primer contains a iso-dC (red base) covalently linked to a fluorochrome. In
the absence of a target, the fluorochrome emits fluorescence (glowing green). In the presence of target, the iso-dG, which is covalently
linked to a quencher, becomes incorporated opposite the iso-dC and quenches its fluorescence (Plexor). D. The 5-end of the primer
has a blocker(red diamond) and a quencher (black) and fluorochrome (green) linked by a target-specific probe sequence and a stem
structure. Priming from the 3-end results in an amplification product which on cooling is targeted by the probe sequence in a
unimolecular reaction (glowing green)(Scorpions). E. A single fluorochrome (green) is quenched by its surrounding ss DNA sequence,
which is also blocked at its 3-end to prevent extension (red diamons). Upon hybridisation, the fluorochrome is dequenched (glowing
green).(e.g. Hybeacons). F. In addition to the single labelled probe, DNA binding-dyes (blue) are added. During the annealing step both
probe and dyes bind to target template and FRET from one to the other (glowing green) can be detected (Resonsense). G. A dual label
(green/black) on a ss probe will be quenched due to the oligonucleotide assuming a random coil formation. Upon hybridisation,
fluorochrome and quencher are separated and fluorescence is emitted (glowing green). H. A single fluorescent label is at the 5- (green)
and 3- (red) ends, respectively, of two probes that target adjacent sequences. Following hybridisation, fluorescence emission due to
FRET is detected (Lightcycler probes). K. Two complementary oligonucleotides form a doublestranded structure, with one labelled
with a fluorochrome at its 5-end and the other with a quencher at its 3-end. Following a denaturation step, the two strands of the
oligonucleotide duplex are separated. Upon annealing in the presence of target, the quencher-labelled strand is displaced by the target
and the fluorochrome emits fluorescence. L. A probe with terminal complementary sequences forms a hairpin structure with
fluorochrome (green) and quencher (black) physically close together. Upon hybridisation, the arms open and fluorescence is emitted
(Molecular Beacons). M.A dual labelled probe binds to its target and is hydrolysed by the nuclease activity of Taq polymerase
(TaqMan ). N.The 5end of the primer contains an antisense DNAzyme binding and active site (blue/red/blue). Upon replication, the
active site is created and cleaves the universal probe at its cleavage site (blue diamond) (Qzyme). O. Same as N, except that the
DNAzyme is made up of two components (MNAzyme).
Figure 1. Selection of
qPCR chemistries.
Protein-targeted PCR
The power of PCR technology has been extended to permit the detection of proteins.
Immuno-PCR
One of the main questions arising from PCR-, especially RT-PCR based results is how any nucleic
acid quantification relates antibodies to detect the to protein expression. Traditionally, this has
required the use of relevant proteins using western blots, immunohistochemistry or
immunoassays. Immunoassays such as the enzyme-linked immunosorbent assay (ELISA) have long
been the mainstay of protein quantification and are widely used in microbiological diagnostics, where
their power lies in their ability to identify pathogens directly by detecting pathogen-specific proteins
as well indirectly by detecting antibodies produced against them. However, despite its specificity, the
use of ELISAs can be limited by their lack of sensitivity. Hence the idea of combining the advantages
of ELISAs and the PCR to create a powerful and versatile method for the detection of low quantities
of protein antigens as well the antibodies that are generated against those antigens. That technique is
called immuno-PCR (iPCR) and has been around for 20 years
[26]
(Figure 2).
iPCR represents an inversion of conventional ELISA protocols: whereas ELISA uses antibodyenzyme
conjugates with the enzymes substrate added subsequently as a freely diffusing species, in iPCR the
substrate (the DNA template), is linked to the antibody while the enzyme is added subsequently.
Amplification of the DNA marker through PCR enhances the limit-of detection (LOD) of a given
ELISA by between 100-10 000-fold. Nevertheless, its main drawback is the problem of cross-
reactivity and nonspecific adsorption, which sets the limit for its selectivity. Hence the practical
application of iPCR has been somewhat stifled by complex protocols and problems of background
noise. For example, physically linking antibodies with DNA turns out to be quite tricky, since the use
of streptavidin protein A chimera
[26]
limits its application to direct detection only, whereas the use
of avidin
[27]
leads to the formation of different species of conjugates and results in high background
noise causing reduced sensitivity and reproducibility. However, the last few years have seen major
advances in the development of new linker molecules, new formats, the association of iPCR with
nanotechnology systems and the availability of ready-to-use reagents from commercial providers with
less laborious protocols.
Figure 2. Basic principles of ELISA and iPCR A. In the ELISA a signal is generated by the action
of an enzyme linked to a detection antibody. B. In iPCR the signal is generated by a qPCR
reaction primed by the DNA linked to the detection antibody. In practice, the detection antibody is
biotinylated and liked through streptavidin to a biotinylated DNA.
The method has also been simplified and a universal iPCR protocol based on the in situ assembly of
biotinylated DNA, streptavidin and biotinylated antibody-antigen complexes has become the most
widely used format for research applications
[28]
. It can be used in both direct and sandwich formats,
requires less hands-on time with far fewer washes needed to eliminate carryover contamination and
displays much reduced nonspecific binding. As a result of these and other improvements iPCR has
gained in robustness and by linking up with other methods such as bead technologies or phage display
is beginning to find a broad variety of applications
[29]
. Standardisation is likely to be enhanced by the
use of commercial, tailored reagents and kits solutions for specific analytical tasks. Since DNA, RNA
and protein detection can be carried out using the same qPCR instrument, it is obvious that iPCR
assays can be used to link information on genetic status, miRNA and mRNA expression and protein
levels.
iPCR may be of most use when looking for very low abundant targets in quality control, diagnostics
and analytics applications. For example, multiplex iPCR may be useful for ultrasensitive detection
and quantification of tiny amounts of target antigens when monitoring complex medical response
patterns of individual patients following treatment. Another area which also calls for ultrasensitive
analyses concerns the monitoring of biological compounds such as contaminants in food, either by
toxins or genetically altered protein components. It will also be interesting to see its application in
the field of single cell analysis, with early indication suggesting that this technology may have a role
to play
[30]
. On the other hand, technologies come and go and the proximity ligation assay (PLA) may
well prove to be more robust, sensitive and reliable than iPCR.
Proximity ligation assay (PLA)
Although PLA is similar to iPCR, the big difference between the two is that PLA results depend on
the binding of two, three or more antibodies to a specific target protein. In practice, the antibodies
have been conjugated to different oligonucleotides to form proximity probes. The antibodies
recognise two or more different epitopes on the same specific target protein or can be used to detect
protein complexes. When antibodies concurrently bind to their targets, the oligonucleotides carried by
the proximity probes are physically brought together (into proximity); a connector oligonucleotide
hybridises to the oligonucleotides and acts as a template for their ligation into a full-length molecule
(Figure 3). This results in a chimeric DNA strand that can be amplified and detected by qPCR or
other detection methods, making PLA several orders of magnitude more sensitive than western
blots
[31; 32]
. A further development is its use for the detection of ternary complexes, where three
proximity probes give rise to the amplifiable DNA molecule
[33]
. Most recently the assay has been
modified to allow the detection of small molecules, which had been difficult since their small
molecular structure prevents two antibodies from binding
[34]
. The new assay, termed competitive
immunomagnetic-proximity ligation assay (CIPLA) uses a single antibody to target clenbuterol and
ractopamine competitively, with LODs 10-50-fold lower than ELISAs.
Obviously assay performance is closely linked to the affinity and specificity of the antibody, since
subsaturating amounts of antibody are used to minimise background noise
[35]
. The requirement for
two independent binding events reduces the likelihood of them occurring in the absence of the
specific target protein, and further minimises the background signal from nonspecific or crossreactive
antibody binding, so contributing significantly to the high specificity and sensitivity of PLA
technology
[31; 32; 36]
.
Figure 3. Proximity ligation assay scheme. A The target protein is bound by two proximity probes
and the oligonucleotides are brought in proximity. B. A connector oligonucleotide can hybridise to
both oligonucleotides, creating a template for ligation. C. An antisense primer synthesises a
complementary strand. D. The newly created DNA-molecule can now be amplified by qPCR.
PLA was first described in 2002, and used a DNA aptamer as the protein binding affinity agent
[37]
.
However, the number of available aptamers is limited, hence antibodies are used that have been
functionalised by either direct covalent coupling of an oligonucleotide or noncovalently by incubating
biotinylated antibodies with a streptavidin-modified oligonucleotide
[36]
. PLAs can be carried out in
several formats:
as homogeneous, including multiplex
[38]
, assays where binding, ligation, and amplification occur in
solution, as in qPCR
in a solid-phase format where the target protein is first immobilised on a solid-phase support using
a capture antibody, then detected with a PLA for the captured protein
[39-42]
in situ where the connector oligonucleotide generates a circular DNA strand upon hybridisation to
the paired proximity probes. Following ligation, rolling circle amplification of the circular template,
which remains hybridised to the proximity probes, by phi29 DNA polymerase results in localised
amplification of the ligation product
[43; 44]
. This technology has been commercialised as Duolink by
Olink Bioscience.
One word of caution: a recent publication shows that various non-linear effects in the in situ PLA
reaction make it a semiquantitative measure of protein co-localisation and suggests that caution
should be exercised when interpreting PLA data in a quantitative way
[45]
. Nonetheless, the potential
of this method is huge with obvious applications in diagnostics, where it has been shown to improve
the accuracy of pancreatic cancer diagnosis
[46]
and in personalised medicine in general
[47]
.
Life Technologies have commercialised a hydrolysis-probe-based PLA assay that expresses results
in terms of the familiar Cq. However, whilst Cq levels obtained from RNA targets are normalised
against validated internal reference genes to account for sampling variation, there are currently no
suitable endogenous controls available for qPCR-based LPAs. Hence Cqs are normalised to total cell
count or total protein concentration. A plot of Cq values against total cell count results in a sigmoidal
curve, in contrast to the straight-line plots typically derived from a nucleic acid dilution series. This
is because Cq values are not just a result of the qPCR component, but are also influenced by probe
binding and ligation events. Furthermore, because the slope of the linear range of hydrolysis-probe-
based PLA assays depends on multiple kinetic components, the slope of a dilution series may vary
from sample to sample.
Conclusion
In the 30-odd years since the first paper demonstrating its practical use, PCR has become the
molecular enabling technology par excellence. It has revolutionised all areas of the life sciences,
medicine, veterinary and agricultural sciences, forensics and many other small niche areas, making it
the most widely used molecular technology today.
However, conventional PCR has several disadvantages: it is an endpoint assay, i.e. target detection
occurs as a separate step after the enzymatic reaction has been completed, involving analysis of
amplification products from the plateau phase of the PCR reaction, where the PCR product is no
longer being doubled at each cycle. Consequently, PCR gel electrophoresis shows broadly similar
amount of product DNA independent of the initial amount of template. That plateau will differ for
each assay due to the different reaction kinetics for each sample; hence this stage is highly
inconsistent between samples and is an important contributor to the frequent lack of reproducibility
and accuracy of conventional PCR data. Conventional PCR is also labour-intensive, subject to
contamination and not easily automated or adapted for high throughput applications. Furthermore,
results are qualitative, and the acquisition of quantitative data using end-point PCR requires the
establishment of additional empirical quantification parameters, e.g. competitive PCR, that vary with
each assay, are tedious to reproduce and are not a trivial matter.
This limitation has resulted in the development of real-time PCR (qPCR), a technology that continues
to revolutionise molecular biology by making it possible to quantify minute quantities of DNA and
RNA with extraordinary speed and precision in a broad range of samples. qPCR can be combined
with a reverse transcription step to quantify RNA or with antibodies to quantify proteins or protein
complexes.
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Chapter 2
Introduction
When asked casually: Who discovered DNA?, most people considering themselves scientifically
literate would probably say Watson and Crick and perhaps also mention Rosalind Franklin. They
might even point at the famous Nature publication of 1953. What a surprise then that the correct
answer is Miescher and the date was 1869.
Johann Friedrich Miescher, a Swiss biochemist, was the first person to isolate a substance that could
be precipitated by acidifying extracted nuclei and re-dissolved when alkaline solutions were added
(1869). These precipitates could not be dissolved either in water, acetic acid, very dilute
hydrochloric acid, or in solutions of sodium chloride, and which thus could not belong to any of
the hitherto known proteins. Since he was certain the substance was derived from the nuclei, he
named it nuclein (1871). He also thought that the presence of nuclein constituted an important
difference between the nucleus and the cytoplasm and suggested that the nucleus function was
dependent on and therefore should be defined by the presence of nuclein. By the way, the first edition
of the book containing Mieschers article ber the chemische Zusammensetzung der Eiterzellen
(Concerning the chemical composition of pus cells) was sold in September 2011 for 7,131.
Mieschers continued work on nuclein led him to conclude that it was a molecule with a high
molecular weight (1872) and to show that it contained carbon, nitrogen and hydrogen and
phosphorous, but no sulphur (1872). He affirmed that it was a multibasic acid (1872),at least a
three basic acid(1874) and thenat least a four basic acid (1874). However, it was difficult to
purify nuclein away from proteins and so most researchers regarded nuclein as related to proteins.
Even when Richard Altman purified the substance free from proteins, he thought he had identified a
novel subcomponent of nuclein, which he called nucleic acid, since it behaved like an acid (1889).
Miescher, on the other hand, was convinced that the two substances were the same. In 1881 Eduard
Zacharias showed that nuclein was an intergal part of the chromosome, thus combining for the first
time the histological concept of chromatin with the chemical substance nuclein.
The four constituent bases, together with the sugar, phosphoric acid and, incidentally, histones, were
identified and named in 1900 by Albrecht Kossel, an achievement that won him the Nobel Prize for
Medicine/Physiology in 1910. In his acceptance speech he noted that the structure and function [of
the cell nucleus] must be associated with the general processes of life.
Nevertheless, the importance of nuclein as a carrier of genetic information was not appreciated for
another fifty years, since it was thought impossible that the complexity of genetic information could be
stored by a molecule made up of four bases. It seemed far more likely that only proteins, made up of
20 or more amino acids, possessed sufficient complexity for this role. The link with the earlier work
by Miescher and others was made in 1944 with the publication of a paper entitled Studies on the
Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types: Induction of
Transformation by a Deoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III" by
Avery, MacLeod and McCarthy, which demonstrated that nuclein, now renamed as DNA was the
likely hereditary substance in bacteria. The role of DNA was corroborated in 1952 with the
publication of a paper by Hershey and Chase Independent functions of viral protein and nucleic acid
in growth of bacteriophage. In it they cautiously state that: protein probably has no function in the
growth of intracellular phage. The DNA has some function.
Despite the lack of knowledge concerning the biological role of nucleic acid, the importance of in
vitro nucleotide synthesis for Chemistry was appreciated from the very beginning, when Emil Fischer
received his 1902 Nobel Prize for the first chemical synthesis of a purine base. Nevertheless, it took
another 50 years (until 1955) for Michelson and Todd to synthesise a dithymidine dinucleotide by
condensing 3-O-acetylthymidine with thymidine 3-(benzyl phosphorochloridate) 5-(di- benzyl
phosphate) and subsequently removing the protecting groups
1]
.
Incidentally, since the synthetic material behaved towards enzymes exactly as the dinucleotidic
fragments obtained by degrading deoxyribonucleic acids, this achievement confirmed the then
postulate of a 3-5 linkage in DNA. The process was cumbersome and slow, with unstable
intermediates, but it provided the launchpad for todays vast oligonucleotide synthesis industry that
can produce large amounts and very pure oligonucleotides as long as 200 bases and even beyond.
They can be produced with a range of modifications that further enhance their usefulness, including
the incorporation of fluorescent dyes and quenchers and specialised nucleotide analogues such as
LNA, that can be incorporated into standard oligonucleotide synthesis using LNA
phosphoramidite monomers.
Phosphodiester to phosphoramidite
Oligonucleotide synthesis depends on protecting reactive parts of the nucleoside molecules until the
polymerisation reaction is started, at which point the protective element is removed and the reaction
can proceed. Carefully controlled reactive elements are continuously cycled from protected to
unprotected, resulting in the linear, step-wise production of an oligonucleotide molecule with a
minimum of undesired reaction intermediates and products. The first complete chemical synthesis of a
gene was described in the early 1970, used the phosphodiester method and resulted in the 77
nucleotide yeast alanine transfer RNA,
[2-4]
. The main disadvantage of this method was the large
numbers of unwanted side chains on each molecule that had to be removed by time-consuming
purification steps. This problem was partly solved by the introduction of two improved synthesis
procedures:
the phosphotriester method, which was characterised by fewer side-chain reactions,
increased stability of reaction intermediates and quicker reaction steps
the phosphate triester chemistry that phosphochlorodites, which improved the synthesis
[5]
.
introduced more reactive nucleoside rate and efficiency of oligonucleotide
The final step towards efficient oligonucleotide synthesis was the replacement of a chloride group
from the phosphochlorodite with an amine group to generate phosphoramidites, which are still being
used today
[6-8]
.
Solid supports
Modern oligonucleotide syntheses are carried out using automated solid-phase methods carried out on
a solid support (resin) held between filters, in columns that enable all reagents and solvents to pass
through freely. Resins are insoluble particles, usually controlled pore glass (CPG) or macroporous
polystyrene (MPPS) and assembled oligonucleotides remain covalently attached to the solid support
material via their 3'-terminal hydroxy group.
Controlled-pore glass is rigid, non-swelling and has deep pores in which oligonucleotide synthesis
takes place. The length of oligonucleotide being synthesised determines the pore size, with 50 nm
pores used for short oligonucleotides up to about 40 bases in length. Since the growing
oligonucleotide blocks the pores and reduces diffusion of the reagents through the matrix, longer
oligonucleotides require larger pores, with 100nm pores used for the synthesis of oligonucleotides up
to 100 bases in length, and 200 or even 300nm pores for longer ones.
MPPS used for oligonucleotide synthesis is a highly cross-linked, low-swelling polystyrene obtained
by polymerisation of divinylbenzene, styrene, and 4-chloromethylstyrene in the presence of a
porogeneous agent. The main advantage of highly cross-linked polystyrene beads is that they
efficiently exclude moisture and allow very efficient oligonucleotide synthesis, particularly on small
scale.
Solid-phase synthesis has several advantages over solution synthesis:
reactions can be quickly driven to completion by using large excesses of solution-phase reagents
since impurities and excess reagents are washed away, no purification is required after each step to
remove unwanted residual reagents
the process is easily automated on computer-controlled solid-phase synthesisers.
Phosphoramidite synthesis
Deoxyribonucleoside phosphoramidite synthesis comprises four main stages that are repeated
cyclically to add each new specific nucleoside to the growing oligonucleotide chain. Synthesis
proceeds in the 3- to 5-direction, with one nucleotide added per synthesis cycle, which consists of
the four steps shown in Figure 4.
Step 1: De-blocking (detritylation)
At the start of the oligonucleotide synthesis the first deoxyribonucleoside, A, G, C or T dependent on
the nucleoside at the 3-end of the desired oligonucleotide, is pre-attached to the resin. It is protected
at its 5-hydroxyl position with a 4,4-dimethoxytrity group which must be removed before a second
base can be added. The exocyclic amines of the bases also have protecting groups attached and the
phosphorous atom is protected with beta-cyanoethyl and diisopropylamine.
After a wash with acetonitrile to remove all traces of acid and reduce adventitious water, the DMT
group is removed with a solution of an acid, such as 2% trichloroacetic acid (TCA) or 3%
dicholoracetic (DCA), in an inert solvent e.g. dichloromethane or toluene. This results in the
formation of an orange-coloured DMT cation that absorbs in the visible region at 495 nm and is
washed away; its yield is measured colourimetrically to help monitor the stepwise coupling
efficiencies of the synthesis reaction. The solid support-bound oligonucleotide precursor now bears a
free 5'-terminal hydroxyl group, which is the only reactive nucleophile on the base monomer and
ensures that the next base can only react with that site.
Step 2: Base Condensation
Since deoxyribonucleoside phosphoramidites are fairly stable and become reactive only upon
protonation, the next base monomer cannot be added until it has been activated. This is achieved by
adding excess acidic azole catalyst, e.g. tetrazole, to the deoxyribonucleoside phosphoramidite. It
protonates the diisopropylamino group of the phosphoramidite, converting it to a good leaving group.
The pKa of this acid is sufficiently high so that it does not remove the DMT group from the reagent,
yet it is still sufficiently acidic to activate the phosphoramidite. The activated phosphoramidite is
added in 1.5 - 20-fold excess over the support-bound material to the synthesis reaction. The
protonated leaving group is rapidly displaced by attack of the 5-hydroxyl group of the support-bound
nucleoside, and a new phosphorus-oxygen bond is formed, creating a supportbound phosphite
triester
[9]
. This occurs very rapidly (20 sec onds) and efficiently (>99%). This reaction is highly
sensitive to the presence of water and is commonly carried out in anhydrous acetonitrile, a good
solvent for nucleophilic displacement reactions. The excess is lower with larger scale syntheses,
which also use higher concentrations of phosphoramidites. Extra tetrazole, unbound base and by-
products are washed away from the reaction column.
Figure 4. The
phosphoramidite oligodeoxynucleotide synthesis cycle
Step 3: Capping
Efficiency of coupling is a critical parameter, since the cumulative effect of a series of poor
couplings results in a poor overall yield of the desired oligonucleotide and in a product that is rather
difficult to purify. This is easily envisaged if one assumes an efficiency of 50% per synthesis cycle.
Following cycle one, only half the oligonucleotides have added the second base. Following cycle
two, only 25% of oligonucleotides would have all three bases, following cycle three only 12.5% of
oligonucleotides would be complete and very soon there would be virtually no full lengthproduct
present. Small differences in theoretical efficiency (Y = (E)
n-1
where (E) is average coupling
efficiency and n is the number of bases in the oligonucleotide) have a significant effect on final yield
of oligonucleotide.
Even the most efficient chemistry, the most sophisticated instrumentation and the purest reagents
cannot achieve a 100% coupling efficiency; instead anything above 98% is readily achievable and
average stepwise yields above 99% can be attained, provided reagents are pure and anhydrous.
Nevertheless, coupling efficiency varies for each base both by type and position in the growing
oligonucleotide, with the frequency of truncated nucleotides at the 3'-end much higher than at the 5'-
end
[10]
. As a consequence, after every cycle of activation and coupling there will be around 1-2%
unreacted 5-hydroxyl groups on the resin-bound nucleotide chain. These need to be inactivated since
they would otherwise take part in subsequent coupling steps, generating a series of deletions in
addition to full length oligonucleotide
[11]
. This is minimised by carrying out a capping step after the
coupling reaction that blocks the unreacted 5-hydroxyl groups. An electrophilic mixture of acetic
anhydride and N-methylimidazole (NMI), dissolved in tetrahydrofuran with the addition of a small
quantity of pyridine, rapidly acetylates the 5-hydroxyl groups, rendering them inert to subsequent
reactions. The pyridine maintains a basic pH that prevents detritylation of the nucleoside
phosphoramidite by the acetic acid formed by reaction of acetic anhydride with NMI. Capped
oligonucleotides remain as short species that are easily removed by a variety of purification methods.
Not all unreacted molecules are capped and continue to participate in subsequent cycles of synthesis,
resulting in near full-length molecules that contain internal deletions, the so-called (n-1)mer species.
Although these molecules will usually work for PCR purposes, they can cause problems with
specificity and should be removed by PAGE or HPLC if primers will be used in multiplex reactions.
Capping also removes any products that may have arisen from reactions of activated
phosphoramidites with the O6 modification of guanosine. These can undergo depurination during the
subsequent oxidation step, with any apurinic sites readily cleaved during the final deprotection of the
oligonucleotide, resulting in shorter oligonucleotides and reducing the yield of full-length product.
Extra acetic anhydride or N-methylimidazole are removed from the column by washing.
Step 4: Oxidation
The newly formed trivalent phosphite triester linkage formed in the coupling step is acid unstable and
must be converted to a more stable species before the next synthesis cycle. This is achieved by iodine
oxidation in the presence of water and a weak base such as pyridine. This forms an
iodinephosphorous adduct that is hydrolysed to yield pentavalent phosphate triester, essentially a
normal DNA backbone protected with a 2-cyanoethyl group, which blocks undesirable reactions at
phosphorus during subsequent synthesis cycles. This oxidation step usually completes one cycle of
oligonucleotide synthesis, although some DNA synthesisers include a second capping step after
iodine oxidation to dry the resin, since any residual water from the oxidation mixture can inhibit the
next coupling reaction. The excess water reacts with the acylating agent to form acetic acid, which is
washed away
Post Synthesis
The 3 -end of the oligonucleotide is attached to the solid support by succinyl linker, which is
unaffected by all the reagents used in the solid-phase oligonucleotide assembly, but is cleavable at the
end of the synthesis. Cleavage is by treatment with concentrated ammonium hydroxide at 55C for 16
hours, which also deprotects the phosphorous by -elimination of the cyanoethyl group, and removes
the acetyl capping groups and the base protecting groups. The resulting aqueous solution, contains a
crude mixture of product oligomer, truncated failure sequences with free 5-hydroxy ends, byproducts
of deprotection and silicates from hydrolysis of the glass support, with more impurities accumulating
as oligonucleotide length increases.
At this point, oligonucleotides are usually desalted, a misnomer since no salt is used during
oligonucleotide synthesis. Instead desalting is a process that removes organic impurities such as
benzamide and acrylonitrile and small molecule impurities such as protecting groups and short
truncation products using gel filtration or organic phase extraction methods. Use of such
oligonucleotides without further purification is cheap and is acceptable for the short primers used in
routine PCR assays. However, truncation species can interfere with multiplex reactions or when
amplicon-length oligonucleotides are needed. Hence, it is important to be aware that a desalted
oligonucleotide includes a significant amount of unwanted material and that if optimal performance is
required, additional purification methods should be used. Typically, polyacrylamide gel
electrophoresis (PAGE) is used to separate the oligonucleotides by size, such that only those
containing the correct number of nucleotides are selected for further use. High performance liquid
chromatography (HPLC) is useful when oligonucleotides contain modified bases, as it separates
oligonucleotides based on charge and/or hydrophobicity. After oligonucleotides have been purified it
is prudent to characterise their quality, especially when synthesizing dual labelled probes or very
long oligonucleotides. This is most easily done by obtaining the molecular mass of the
oligonucleotide by recording its mass spectrum. This can be done either by electrospray mass
spectrometry (ES MS) or by matrix-assisted laser desorption/ionisation time-of-flight mass
spectrometry (MALDI-TOF).
Conclusions
Despite the basic principles being the same since 1981, continuous modifications and improvements
to reagents and equipment is resulting in the synthesis of ever-longer, pure and inexpensive
oligonucleotides. There are also new methods being proposed that may, ultimately, result in a
challenge to the undisputed superiority of the currently supreme phosphoramidite method. The uses
for oligodeoxynucleotides are expanding all the time, and there is increasing interest in the synthesis
of ribonucleotides, driven by the discovery of small noncoding RNAs and the practical applications
of RNA interference. We have come along way since the synthesis of a dimer in 1955, with synthesis
of a 100-mer routine and a 200-mer not impossible. And all of this while prices are at rock bottom.It
is certainly reassuring to know that of all the components of a PCR assay, the synthesis of an
oligonucleotide is the least likely building block to cause a problem.
References
1. Michelson A.M., Todd, A. R. (1955) Nucleotides part XXXII. Synthesis of a dithymidine
dinucleotide containing a 3':5'-internucleotidic linkage J. Chem. Soc. 2632-2638
http://pubs.rsc.org/en/content/articlelanding/1955/jr/jr9550002632
2. Khorana, H. G., Buchi, H., Caruthers, M. H., Chang, S. H., Gupta, N. K., Kumar, A., Ohtsuka, E.,
Sgaramella, V. and Weber, H. (1968) Progress in the total synthesis of the gene for alatRNA. Cold
Spring Harb Symp Quant Biol 33:35-44
3. Agarwal, K. L., Buchi, H., Caruthers, M. H., Gupta, N., Khorana, H. G., Kleppe, K., Kumar, A.,
Ohtsuka, E., Rajbhandary, U. L., Van de Sande, J. H., Sgaramella, V., Weber, H. and Yamada, T.
(1970) Total synthesis of the gene for an alanine transfer ribonucleic acid from yeast. Nature 227:27-
34
4. Khorana, H. G., Agarwal, K. L., Buchi, H., Caruthers, M. H., Gupta, N. K., Kleppe, K., Kumar, A.,
Otsuka, E., RajBhandary, U. L., Van de Sande, J. H., Sgaramella, V., Terao, T., Weber, H. and
Yamada, T. (1972) Studies on polynucleotides. 103. Total synthesis of the structural gene for an
alanine transfer ribonucleic acid from yeast. J Mol Biol 72:209-217
5. Ohtsuka, E., Ikehara, M. and Soll, D. (1982) Recent developments in the chemical synthesis of
polynucleotides. Nucleic Acids Res 10:6553-6570
6. Beaucage, S. L. and Caruthers, M. H. (1981) Deoxynucleoside phosphoramiditesA new class of
key intermediates for deoxypolynucleotide synthesis Tetrahedron Letters 22:1859-1856
7. Caruthers, M. H., Beaucage, S. L., Becker, C., Efcavitch, J. W., Fisher, E. F., Galluppi, G.,
Goldman, R., deHaseth, P., Matteucci, M., McBride, L. and et, a. (1983)
Deoxyoligonucleotide synthesis via the phosphoramidite method. Gene Amplif Anal 3:1-26
8. McBride, L. J. and Caruthers, M. H. (1982) An investigation of several deoxynucleoside
phosphoramidites useful for synthesizing deoxyoligonucleotides Tetrahedron Letters 24:245-248
9. Berner, S., Muhlegger, K. and Seliger, H. (1989) Studies on the role of tetrazole in the activation
of phosphoramidites. Nucleic Acids Res 17:853-864
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC331708/pdf/nar00212-0033.pdf
10. Temsamani, J., Kubert, M. and Agrawal, S. (1995) Sequence identity of the n-1 product of a
synthetic oligonucleotide. Nucleic Acids Res 23:1841-1844
11. Hecker, K. H. and Rill, R. L. (1998) Error analysis of chemically synthesized polynucleotides.
Biotechniques 24:256-260
Chapter 3
Introduction
The previous narrative rather implies that the PCR is a fairly simple process and only requires a
combination of a DNA template, two oligonucleotide primers, a dNTP mix, a simple buffer (50 mM
KCl, 1.5 mM MgCl
2
, 10 mM Tris-HCl, pH 8.3), a thermostable polymerase and a thermal cycler to
allow the amplification of any known DNA target. As is often the case, this is true, but it is also
completely wrong. Nature favours simplicity, and since PCR is nature in action, it is not surprising
that its concept is not simply ingenious, but also simple. However, nature is not bent on absolute
specificity and makes use of mistakes that occur during replication to increase diversity. In addition,
all enzymatic reactions always proceed under optimal conditions and a polymerase never pauses to
count copy numbers before proceeding with its synthesis. Most obviously, cells replicate and repair
their nucleic acids without having to denature them.
In vitro , on the other hand, this is clearly not the case and the success of a PCR reaction depends on
a fine balance between these components, any one of which can change the outcome of a PCR
reaction. These must be chosen according to the aims of the individual PCR experiment, individually
optimised and balanced with all other components of the reaction setup. Hence it is worth reviewing
these in some detail, since understanding their contributions will help with initial PCR assay design
and optimisation as well as later troubleshooting.
DNA template
A PCR reaction must cope with a wide range of target template copy numbers that can range from
vast numbers, e.g. tens of millions during an acute viral infection to samples with zero copies of
target but vast numbers of other DNAs. In general, it is advisable to add as little DNA as is feasible
to a PCR assay, since too high a concentration may lead to poor results due to inhibition of and
mispriming by the DNA polymerase. However, this can pose an obvious problem when the aim is to
amplify very low copy number targets since the target DNA template will comprise but a small
percentage of the total DNA in a sample. Conversely, when a sample is made up of very dilute DNA,
amplification may be impeded by its adsorption to plasticware, by the increased risk of degradation
or by non-specific primer annealing resulting in false positive results. In addition, contamination
poses a constant threat.
Technical issues such as DNA quality, which refers to purity (i.e. the absence of inhibitors) as well
as integrity have a significant effect on the reliability of a PCR reaction
[1]
and must be addressed
using rigorous standard operating procedures. DNA extraction procedures affect DNA quality
[2]
and
extracting DNA from a tissue culture cell is obviously not comparable to extracting DNA from an
environmental sample containing bacterial or fungal spore. One is rather straightforward; the other
has the serious potential for a failed or inefficient DNA extraction leading to the reporting of a false
negative result.
Inhibition
Compounds that inhibit Taq polymerase enzyme activity are potentially present in many samples:
haeme can inhibit PCR amplification of target DNA in samples containing blood
[3, 4]
. Faeces have
long been a valuable reservoir for PCR analysis
[5]
, yet the breakdown products of haeme, such as
bilirubin, as well as bile salts can inhibit the PCR
[6]
. In addition, many of the reagents used to
cultivate microorganisms, to stain cells or to prepare samples for PCR can inhibit the reaction
[7, 8]
.
Many sources of inhibition are chemically ill defined; e.g. humic substances are a mixture of complex
polyphenolics produced during the decomposition of organic matter, are ubiquitous in soil and water
and may be co-purified with any material obtained from environmental samples
[9]
. DNA extraction
from plants has to cope with these as well as with polysaccharides, which form complexes with and
become bound to the DNA and inhibit Taq polymerase
[10]
. Finally, it has been known for a very long
time that components of the reverse transcription reaction are important sources for impurities that
inhibit the PCR reaction following cDNA synthesis
[11-15]
.
These are just a few in a long list of components that may act as, sometimes inadvertent, PCR
inhibitors or enhancers, and directly affect PCR results. Critically, it appears that different PCR
reactions have differential susceptibility to inhibitors
[16]
. An assessment of inhibitors copurified
during the extraction of DNA from urine revealed that susceptibility to inhibition was highly variable
between reactions. There was no obvious explanation why one reaction should be more susceptible
to inhibition than another, although a possible association with amplicon GC content was noted. This
has serious implications for any PCR-based gene expression studies, including those using PCR
arrays, as well as for PCR-based molecular diagnostic assays. It is not safe to assume that different
PCR reactions are equally susceptible to inhibition by substances co-purified in nucleic acid extracts
and it is essential to perform routine quality checks on all samples, particularly if they have been
extracted from anywhere but a tissue culture environment.
Integrity
The integrity of the target sequence is another important parameter that affects the accuracy of qPCR
assays and is often overlooked when targeting DNA. DNA damage is not always predictable and can
result in false negative results, particularly critical when quantitative data are used in a clinical
setting. The importance of correctly assessing DNA integrity is emphasised by the contradictory
results obtained when assessing DNA integrity as a biomarker for monitoring minimal residual
disease or response to therapy in cancer. Some reports suggest that plasma DNA integrity may be
increased in cancer patients
[17-19]
or associated with therapy response in breast cancer
[20]
, whereas
others find no such evidence
[21, 22]
.
Quantitative results depend on the number of intact, amplifiable target sequences in the sample, which
can be affected by DNA damage that occurred in vivo or during sample collection, transport, storage,
and processing. In most cases there is probably insufficient DNA damage to interfere with qPCR
results, but this is not so for some conditions, e.g. DNA extracted from stained microscope slides or
from archival material. One way to determine the proportion of target sequences that are amplifiable
is to use a method that assumes that if the DNA lesions preventing amplification occur randomly, then
the Poisson distribution will describe their number in a given length of DNA. The mean number of
lesions per base then provides a simple measure of DNA integrity, allowing the calculation of he
amplifiable fraction of a target sequence from this number and the target length
[23]
.
Extraction efficiency
The use of spike-and-recovery controls can be useful for identifying concerns regarding extraction
efficiency although, as always, it is important to choose appropriate ones. The surrogate must be
absent from the native sample, co-concentrate with the target of interest, lyse with equal effectiveness
compared to target cells, and contain DNA that is extracted and recovered with efficiency equivalent
to that of targeted cells
[24]
. Since this can be difficult to achieve, some researchers add naked spike
DNA to their sample just prior to extraction; however, this may become degraded and lead to
inaccurate and variable evaluation of DNA extraction efficiency. Conversely, if the spike is added
following the extraction process, it can no longer serve as an extraction control, although it can be
used for monitoring of inhibition. Once extracted, detection becomes an issue, since an additional
primer pair will be required to detect the spike. If carried out as a separate reaction, further cost is
incurred; if carried out as a dual- or multiplex reaction, further optimisation is required with possible
loss of amplification efficiency.
Primers
Taq polymerase, like all DNA-dependent DNA polymerases
[25]
, requires a preexisting primer from
which to extend DNA synthesis. In PCR reactions, these are usually oligodeoxynucleotides, although
the polymerase can extend primers containing modified bases
[26]
. Primers anneal at temperatures that
are depended on their sequence, concentration, length and ionic environment. Although the shortest
primer length required for extension by Taq polymerase is an octamer
[27]
, longer oligonucleotides
prime more efficiently and most PCR primers are between 18 and 22 nucleotides long.
As described in detail in Definitive qPCR: Assay Design, primers are the single most critical
constituents that determine the success of a PCR reaction. All things being equal, a poorly designed
primer pair will lead to a inferior PCR reaction characterised by poor reproducibility, non-specific
amplification, primer-dimer formation, poor yield and, in extremis, no amplification product at all.
Critical primer attributes such as specificity, annealing temperature and efficiency as well as
potential to form secondary structures are defined by their sequence and length.
Primers should be sufficiently complex to minimise the likelihood of annealing to sequences other
than the chosen target. There is a 1:256 (4
4
) chance of finding a specific four-nucleotide sequence in
any given DNA sequence. Hence, an 18 base sequence will statistically be present only once in every
6.8x10
10
bases, around 20 times the size of the human genome. Each additional nucleotide makes a
primer four times more specific, making the ideal length for a primer around 18-22 nucleotides. If
care is taken during the design process to minimise any 3-complementarity of primers with
mismatched targets and to optimise empirical annealing temperatures and primer concentrations, a
20-24 base primer set will be exquisitely specific for a single target sequence.
Annealing efficiency is the second important hallmark of a first-rate primer, since a small decrease
in annealing efficiency will result in appreciably diminished yield of amplification products
[28]
. The
annealing temperature (Ta) is defined as the temperature where the efficiency of PCR amplification is
maximal without generating non-specific products and is the critical temperature for ensuring an
efficient PCR reaction. It is often wrongly referred to as a primers melting temperature (Tm), but is
in fact 510C below that. Tm only refers to the temperature at which 50% of the oligonucleotide
target duplexes have formed. It is always worth determining the Ta empirically, since every method
of calculating Tm gives a different result, but in general a Ta range of 55-60C is optimal. It is also
important to make sure that both primers of a pair have approximately the same Ta, since widely
diverging Ta (>about 2C) will result in inefficient, non-exponential amplification.
Any potential for secondary structure formation must be avoided, since this will dramatically
interfere with the primers ability to anneal to its target and prime the PCR reaction. Closely related,
inter-primer homology between two oligonucleotides present at vast excess compared to their target
will also result in poor annealing and an unreliable PCR reaction. If the homology is at the 3-end of
one primer and anywhere along the length of its partner, primer dimers may form and again curtail
amplification efficiency
[29]
.
Deoxynucleoside triphosphates
Deoxynucleoside triphosphates (not deoxynucleotide triphosphates!) are the normal substrate for
DNA polymerases, and their concentration in a standard PCR reaction is between 50 and 200 M of
each dNTP. Higher concentrations of dNTPs (up to 500 M) can increase DNA yield and result in the
synthesis of longer products. However, increasing the dNTP concentration requires a reoptimisation
of the complete PCR reaction, including an adjustment of the Mg
2+
concentration, since dNTPs and
Mg
2+
form soluble complexes that are the actual substrate that DNA polymerases recognise. Too low
concentrations of dNTPs can result in incomplete strand polymerisation and premature termination of
DNA synthesis during the elongation step of the PCR cycle.
Purity
Although Taq polymerase fidelity is not materially affected by changes to the concentration of dNTPs,
with its base substitution error rate varying only two-fold between 1 mM (1/6,000) and 1 M dNTPs
(1/12,000) at 10 mM MgCl
2
(pH 7.2)
[30]
, successful PCR reactions do require pure and stable
dNTPs. They can be manufactured from deoxynucleoside monophosphates (dNMPs) either by
chemical synthesis involving the addition of inorganic pyrophosphates (PPi) or enzymatic
phosphorylation synthesis. Enzymatic synthesis has the advantage of resulting in fewer impurities that
inhibit the PCR reaction. Nevertheless, an inadequate manufacturing process can leave dNTP
preparation containing PCR inhibitors, including NTPs, other dNTPs, dNMDs and deoxynucleoside
diphosphates (NDPs). dNTPs may also contain macromolecular contaminants such as DNA from
human and bacterial origin, since enzymes of bacterial origin are commonly used during enzymatic
dNTP synthesis and human DNA is ubiquitously present during dNTP handling. Both can give false
positive results. It is also important that the dNTPs are free from residual enzymatic activities,
including DNAse and RNAse that can result in false-negative results and nickase activity that can
affect the amplification template. Chemical synthesis can lead to dNTP solutions that contain critical
concentrations of inorganic PCR inhibitors such as chloride, acetate, pyrophosphates, magnesium,
calcium and heavy metals, inorganic pyrophosphates and tetraphosphates. All of these may decrease
the sensitivity or completely inhibit a PCR reaction, e.g. since PPi chelates Mg
2+
, an excess can lead
to an imbalance of Mg
2+
.
Interestingly, solution nucleotides are purer (>99%) than the lyophilised version (<98%). In addition,
dNTPs are more stable in solution, where they hydrolyse more slowly into NDPs and NMPs than
lyophilised preparations. This can be especially noticeable if dNTPs are kept in the refrigerator,
whereas at -20C, the rate of degradation for both forms is less than 1% per year. The optimal pH for
storage is between pH 7.5-8.2 (pH at 20C); acidic pH leads to hydrolysis of dNTPs, rendering them
less suitable for PCR applications. Since the pH of dNTP Na-salts solutions can differ from the pH at
20C during freezing/thawing cycles, it is best to use Li-, rather than Na-salts solutions. Another good
reason is that dNTPs are more soluble in Li-salts; this prevents particularly dGTP from precipitating
during freezing and so avoids an imbalance in the final dNTP concentration. Finally, dNTPs should
be stored at concentrations of around 100 mM, since significant hydrolysis occurs when they are
stored at low concentrations.
dUTP
A major problem with PCR is contamination from DNA either already present in the laboratory
environment (including the operators skin and hair) or newly introduced through the various steps
involving sample handling, nucleic acid extraction, the PCR itself or post-PCR procedures. This is of
particular concern for diagnostic PCR. Routine precautions to minimise environmental contamination
includes following good laboratory practice, using separate laboratories with laminar flow hoods for
setting up the assay, running it and then analysing the data, keeping numerous aliquots of all reagents
and using fresh ones as often as is feasible, using commercial prepackaged plasticware and wearing
disposable gloves. Carry-over contamination can be minimised by substituting dUTP for TTP in the
dNTP mix, which results in all PCR products containing uracil instead of thymine
[31]
. During the
setup of subsequent PCR reactions, thermolabile Uracil-Nglycosylase (UNG) is added to the reaction
mixture together with a short pre-PCR incubation step. This hydrolyses the N-glycosidic bond
between the deoxyribose sugar and uracil of any previously amplified DNA that might contaminate
the new assay.
Enzymes
DNA-dependent DNA polymerases are classi fied into families based on sequence similarity
[32, 33]
,
with the most important enzymes for PCR in families A and B. Taq and Bst are family A repair (not
replicating) polymerases involved in excision repair and processing of Okazaki fragments generated
during lagging strand synthesis. Most archaebacterial polymerases are grouped in family B and
include both repair and replication enzymes. Despite their diverse biological functions the structural
and chemical mechanisms of base incorporation by the polymerases in these two families seem to be
very similar
[33]
.
DNA synthesis
Polymerases synthesise DNA in repeated cycles by moving along a partly single stranded, partly
double-stranded DNA chain in a 53' direction
[34]
. Although the fidelity of different polymerases
varies, the basic process of DNA synthesis ensures that all DNA polymerases exhibit relatively low
error rates, ensuring that in each catalytic cycle a single nucleotide complementary to the nucleotide
on the template strand is added to the nascent chain of the double-stranded DNA. In order to achieve
this, in vivo polymerisation involves a series of checkpoints:
The first is introduced by the hydrogen bonding that stabilises complementary base interactions
during the formation of a base pair. This stabilisation cannot occur if a noncomplementary base is
placed opposite the template base.
A second level of fidelity is provided by certain amino acids forming hydrogen bonds with the
minor-groove side of the base pair in the active site. This interaction occurs only if a correctly
spaced base pair has formed in the active site.
A third level of fidelity is achieved by the DNA polymerases undergoing a conformational change
around an incoming dNTP. The finger domain rotates and forms a snug pocket into which only a
correctly shaped base pair will fit.
Many polymerases have additional means of improving the fidelity of DNA synthesis involving
53 and/or 35 exonuclease activities. The 53 activity corrects errors in pre-existing DNA
and removes RNA primers used for DNA replication. Cleavage can occur at a bond several residues
from the 5-end and this activity is crucial for repairing damaged DNA. The 35 activity provides
a proofreading function
incorporated into the DNA chain.
and immediately removes any wrong base mistakenly
The exonuclease domain does not participate in the polymerisation reaction but is activated when the
end of a growing strand containing an incorrectly incorporated nucleotide leaves the polymerase site
and transiently moves to the exonuclease site. The exonuclease activity then hydrolyses any
mismatched nucleotide from the 3 end of DNA.
Types of polymerase
In vitro , for any given enzyme, fidelity during the PCR reaction is largely determined by its intrinsic
error rate under the reaction conditions employed and can be minimised by optimising pH, Mg
2+
and
nucleotide concentrations
[35, 36]
. It is also possible to use DNA polymerase blends, depending on the
length of the target and the accuracy of the polymerisation required
[37]
. There are numerous
thermostable DNA polymerases available for PCR, making it important to choose the appropriate one
for whatever task is on hand. Like Taq DNA polymerase, DNA polymerases from the genus Thermus,
such as Tth
[38, 39]
and Tfl
[40]
, have a 53 exonuclease DNA repair activity, but no 35
proofreading activity. Other eubacterial thermophilic polymerases include Bst
[41]
, Bca
[42, 43]
, Tru
[44]
and Tsp
[45]
.
Another source of thermostable polymerases is provided by the archaebacteria, a group of bacteria
distinct from eubacteria
[46]
that include extreme thermophiles capable of growth up to 100C
[47, 48]
.
Their polymerases are assigned mostly to the B family of DNA polymerases, with sequence similarity
to human DNA polymerase A
[49]
and E. coli DNA pol II and comprise a range of additional
heatstable enzymes with a performance often superior to that of Taq polymerase
[50, 51]
.
Thermococcus litoralis (Tli) polymerase possesses exceptional thermostability, with an activity half-
life of 8hr at 95C and 2h at 100C
[52]
. It synthesises DNA at a rate of 67 nucleotides/second, but has
very low processivity (7 nucleotides per binding event). It has a 35, but no 53 exonuclease
activity. Another such enzyme is the Pfu DNA polymerase from Pyrococcus furiosus
[53]
, which
retains around 95% of its initial activity after 1h at 95C and also has a 35 proofreading activity,
but no 53 exonuclease activity
[47]
. Other, albeit less extremely thermostable polymerases include
those from Sulfolobus acidocaldarius (Sac)
[54, 55]
, S. solfataricus (Sso)
[56]
, Thermoplasma
acidophilum
[57]
and various others
[58]
.
The 3 5 activity of these enzymes confers an additional benefit on these enzymes. They display
significantly higher fidelity than Thermus-derived polymerases, expressed as the ratio of the error
rate (number of misincorporated nucleotides per base synthesised) over the number of amplicon
doublings, defined as the ratio of amplicon yield over amount of input target DNA. Whereas error
rates of Taq polymerase range from 5x10
-4
to 2.1x10
-6[30, 59-61]
, archaeal polymerases have
significantly lower error rates at 7x10
-5
to 8x10
-6
per doubling
[52, 53, 62-66]
.
A major limitation of Taq polymerase is its inability to amplify damaged DNA, an important
consideration for forensic applications or amplification from ancient or formalin fixed DNA. Dpo4
from S. solfataricus, and related enzymes from Y-family DNA polymerase possess a larger and less
constricted active site that can accommodate bulky or non-coding adducts. As a result, these
polymerases can bypass DNA lesions such as abasic sites, cissyn cyclobutane pyrimidine dimers,
pyrimidine 6-4 pyrimidone at a dithymine site, cis-Platin and N-acetyl-2-aminofluorene guanine
adducts. Such properties make these enzymes ideally suited for the PCR amplification of damaged
DNA samples, ideally as a blend of Taq and Dpo4-like enzymes
[67]
.
The initial drawback of these enzymes, their slow-ish polymerisation rate of 9-25 bases/second vs
the 47-61 recorded for Taq polymerase was solved by the discovery of Thermococcus
kodakaraensis, as its polymerase KOD1 proceeds at 106-138 nucleotides/second
[51]
. This type of
polymerase allows the development of ultrafast protocols using novel instrumentation, for example
the substitution of metal heating blocks by liquid for reaction times of 94 seconds for 35 cycles
[68]
.
Other extreme thermophiles also harbour enzymes that have significantly improved processivity
compared with Taq polymerase. TNA1_pol, isolated from Thermococcus sp. NA, has a half-live of
3.5 h at 100C and 12.5 h at 95C, an extension rate that is similar to Taq polymerases at 60
nucleotides/second but a processivity of 150 nucleotides, which is two-three times that of Taq
polymerase
[69]
.
A second problem with these enzymes was that they are prone to dUTP poisoning. Deamination of
dC to dU is the most common mutagenic change in DNA and if not repaired prior to replication,
directs incorporation of dA, generating a G:C to A:U transition mutation in half the progeny. Native
family B DNA polymerases from archaea recognise the presence of unrepaired U in a template
strand, stall DNA synthesis
[70]
and so cannot copy uracil-containing templates
[71]
. This biologically
important property has a severely negative effect on enzyme performance during a PCR reaction
because heating enhances dCTP deamination to dUTP, with longer cycling times initially used due to
the low processivity exacerbating the problem
[65]
. This problem was first addressed by the
development of an exonuclease deficient KOD1
[72]
as well as identification of a thermostable
dUTPase and its incorporation into the reaction buffer, which significantly enhanced the performance
of these polymerases
[73]
. Better even, the identification of a pocket in the N-terminal domains of
archaeal DNA polymerases that discriminates dU allowed the engineering of a Pfu polymerase that
can read through template-strand dUs
[74]
. More recently, a family B DNA polymerase from
Nanoarchaeota equitans (Neq) has been shown to be capable of amplifying DNA in the presence of
uracil as well as hypoxanthine, the deamination product of adenine
[75]
.
Depending on the application, it is worth remembering that Taq and Tth polymerases can add a single
A to the 3 ends of its polymerisation product without requiring a template (extendase activity),
whereas Tli, KOD and Pwo leave blunt ends
[76, 77]
. As discussed earlier, PCR-based clinical,
environmental and forensic tests are susceptible to inhibition from substances contained within blood,
soil or other samples from which nucleic acids are extracted and it has been known for a long time
that different polymerases are inhibited to different degrees by these components
[78-80]
. Hence
inhibition can be adressed to some extent by choosing the most appropriate thermostable DNA
polymerase
[81]
and there are engineered Taq polymerase mutants available that can tolerate up to
40% whole blood or serum in the reaction, or concentrations of soil which are completely inhibitory
for standard thermostable enzymes
[82]
.
Today, molecular engineering to introduce improved performance has modified the original native
enzymes. The Stoffel fragment of Taq polymerase was the first in a long line of modified
polymerases. It is a modified Taq polymerase lacking the N-terminal domain that harbours the 5-3
exonuclease activity. It was engineered by deleting the first 867 bp of the Taq polymerase gene to
yield a 544 amino acid protein with a molecular weight of 61.3 kDa that is around two times more
heatstable (21 min vs. 9 min half-life at 97.6C, respectively)
[83]
. It is less processive than Taq (5-10
nucleotides vs 50-60), can extend DNA at a rate of around 50 nucleotides/second and works over a
broad range of Mg
2+
concentrations (2-10mM) in lower ionic strength buffer (10mM KCl) which may
be useful for multiplex reactions. Covalently linking the polymerase domain from Taq
[84]
or
Thermococcus pacificus (Tpa)
[85]
to sequence non-specific dsDNA binding proteins, e.g. Sso7d
from S. solfataricus, has been shown to be a useful means of increasing the processivity of the
polymerase without compromising catalytic activity and enzyme stability. As discussed earlier, the
naturally lower error rates of Pfu DNA polymerase have also been reduced even further by selecting
for appropriate mutations, and the processivity of Pfu-based enzymes has been increased by fusing
polymerases with a high affinity double-stranded DNA binding domain that anchors the DNA
polymerase and so reduces dissociation from the DNA template.
Hot start
Although a well-designed PCR reaction is highly specific, with primers annealing to a single target at
their optimal annealing temperature, reactions are set up at room temperature. Furthermore, even
though the reaction tubes are kept on ice, pipetting, spinning and shuttling between ice bucket,
microfuge and thermal cycler leaves plenty of scope for low-level synthesis from general priming.
There is additional opportunity for such generic priming during the heating phase of the first cycle,
before the DNA becomes denatured. This leads to random product formation and primer
oligomerisation. Once such priming occurs, any non-specific products will be amplified efficiently
during the subsequent PCR reaction, leading to reduced yields of the intended amplicon and so
reducing the sensitivity of the experiment.
This gave rise to the idea of using a hot start procedure, which initially involved adding the primers
to the reaction only after the reactants had been heated past the primer annealing temperature. This
was of course not just rather unwieldy but also increased the likelihood of contamination. A slightly
more convenient procedure involved the use of a layer of solid wax to separate the reagents until the
first denaturation step. This melted the wax and convectively mixed the two aqueous layers
[86]
.
Whilst an improvement, it was still far from ideal and led to the idea of an enzyme that is inactive at
low temperatures and become activated after a period of incubation at an elevated temperature. The
first such systems used antibodies against Taq polymerase to inhibit Taq activity below 40C;
following the first heating step, the antibodies were also denatured, leaving a fully active Taq
polymerase
[87, 88]
.
An alternative approach used a single-stranded DNA library screen to isolate DNA sequences
capable of inhibiting Taq polymerase at room temperature but not above 40C. Two families of
aptamers were identified, with heterodimeric aptamers resulting from their head-to-tail combination
being the most efficient way of inhibiting Taq activity selectively at 20 to 25C, but allowing Taq to
become fully active above 40C. Although 40C is not ideal, this system has the advantage of a
reversible activation/inactivation
[89]
. A similar, albeit less convenient approach uses double stranded
DNA fragments to achieve inhibition of Taq polymerase
[90]
. A fourth mechanism blocks the
polymerase through chemical modification, which is unblocked during the first heating step. Yet
another approach makes use of cold-sensitive mutants such as Cesium Taq and Cesium KlenTaq,
which have markedly reduced activity at 37C yet retain apparently normal activity at 68C and
resistance at 95C
[91]
.
Reaction mixes
Modern reaction buffers are aqueous solutions that have a highly stable pH even if there is
evaporation and have progressed way beyond the simple buffers used in the early days of PCR, when
catalogues listed the buffer contents. Certain templates can be difficult to amplify, resulting in non-
linear amplification with little or no specific product or a range of non-specific products. Hence most
commercial buffers contain co-solvents, which can influence template melting properties, PCR
enhancers that increase product yield and compounds that coat the sides of the PCR tubes so that
reagents are not lost through adsorption to the tube walls.
Additives
GC-rich regions can form rigid, constrained secondary structures that are dif ficult or impossible for
the DNA polymerase to enter under standard PCR conditions. Organic additives that can improve
results include dimethylsulfoxide (DMSO)
[92]
, formamide and glycine
[93]
, the amino acid analogue
Betaine (N,N,N,-trimethylglycine) obtained from sugar beet
[94]
, low molecular weight sulphones
chemically related to DMSO such as sulfolane and methyl sulfone
[95]
and tetramethylene sulfoxide
[96]
and low molecular weight amides e.g. acetamide
[97]
. DMSO and formamide are thought to aid in the
amplification of GC-rich templates in a similar manner by interfering with the formation of hydrogen
bonds between the two strands of DNA
[98]
. Both cause inhibition of Taq DNA polymerase and,
presumably, other DNA polymerases as well at concentrations greater than 10% and 5%,
respectively
[99]
. Betaine reduces the amount of energy required to separate the strands of a GC-rich
DNA template
[100]
, eliminates the differences in melting temperature between AT and GC domains,
probably by preferentially binding to AT pairs in the major groove of the B form double helix
[100]
and affecting the local structure of the DNA molecule
[101]
and protects Taq polymerase from
denaturation
[102]
.
The presence of detergent improves the activity of some enzymes, presumably by reducing
aggregation. Thermostable pyrophosphatase (< 1 U/ml) can be added to hydrolyse pyrophosphate
produced during dNTP incorporation. In some cases, general stabilising agents such as BSA
(0.1mg/ml), gelatin (0.11.0%), and nonionic detergents (00.5%) can overcome failure to amplify a
region of DNA. BSA has also been shown to overcome the inhibitory effects of melanin on
RTPCR
[103]
. Nonionic detergents, such as Tween-20, NP-40, and Triton X-100, have the
additional benefit of being able to overcome the inhibitory effects of trace amounts of strong ionic
detergents, such as 0.01% SDS
[104]
.
Cations
Monovalent cations are essential for maintaining a high ratio of specific to nonspecific annealing of
the primers as they neutralise the negatively charged phosphate groups on the DNA backbone, weaken
the electrorepulsive forces between the DNA strands and so facilitate the annealing process between
primer and template. Most commercially available PCR buffers contain K
+
as the only monovalent
cation. It binds to the phosphate groups on the DNA backbone and so stabilises the annealing of the
primers to the template. QIAGEN have found that a balanced combination of K
+
and NH4
+
produces
higher yields of specific PCR product obtained over a wide range of annealing temperatures. NH4
+
,
which exists as both ammonium ion and ammonia under thermal cycling conditions, can interact with
the hydrogen bonds between the bases to principally destabilise the weak hydrogen bonds of
mismatched primer template bases. The combined effect of the two cations maintains the high ratio of
specific to nonspecific primer:template binding over a wide range of temperatures.
Like all enzymes with nucleoside triphosphate substrates, DNA polymerases require metal ions,
typically, Mg
2+
, for activity
[105]
. The role of Mg
2+
in PCR is dual: promoting DNA/DNA interactions
and forming complexes with dNTPs that are the actual substrates for thermal DNA Polymerase.
Typically, a plot of PCR yield vs Mg
2+
produces a bell curve with a broad maximum, although its
precise shape varies depending upon the particular enzyme. Different enzymes have different optimal
Mg
2+
ranges: Taq polymerase works best between 14mM Mg
2+
, Tth polymerase between 1.5
2.5mM Mg
2+
, Pfu and Tli polymerases between 26mM, and Tfl polymerase between 14mM Mg
2+
.
Some enzymes perform better with MgSO
4
rather than with MgCl
2
.
If Mg
2+
concentration is too low, primers may fail to anneal to the target DNA and the polymerase
activity may be poor. When Mg
2+
is too high, non-specific amplification can occur more easily due to
the stabilisation of mismatches. The stronger base pairing can also result in the strands of the
amplicon failing to separate completely during the denaturation step. Examination of the structures of
DNA polymerases with bound substrates and substrate analogs reveals the presence of two metal ions
in the active site. One metal ion binds both the dNTP and the 3-OH group of the primer, whereas the
other interacts only with the 3-OH group. The two metal ions are bridged by the carboxylate groups
of two aspartate residues in the palm domain of the polymerase. These side chains hold the metal ions
in the proper position and orientation. The metal ion bound to the primer activates the 3-OH group of
the primer, facilitating its attack on the -phosphate group of the dNTP substrate in the active site.
The two metal ions together help stabilise the negative charge that accumulates on the pentacoordinate
transition state. The metal ion initially bound to dNTP stabilises the negative charge on the
pyrophosphate product.
Mg
2+
also affects polymerase fidelity, which responds not to absolute Mg
2+
concentration, but rather
to the amount of Mg
2+
in excess over the total deoxynucleoside triphosphates present during DNA
synthesis.
[30]
. Hence although template DNA concentration, chelating agents present in the sample and
the presence of proteins can affect the amount of free magnesium in the reaction, dNTP concentration
is the most critical component that determines the concentration of free magnesium. Consequently,
small increases in dNTP concentrations can rapidly inhibit the PCR reaction and any increase in
dNTP concentration requires an increase in the concentration of Mg
2+
ions in order for the reaction to
work optimally
[106]
.
Specialist reagents
Accurate and reliable discrimination of sequences that differ by a single nucleotide constitutes one of
the major challenges for any molecular targeting systems. In general, the shorter the probe, the greater
the effect of a single mismatch on a probes hybridisation profile; hence the use of high affinity
analogues such as LNA
[107]
and minor groove binders
[108; 109]
to generate fluorescent probes that are
shorter than standard DNA-based equivalents and achieve better performance
[110]
. Other approaches
include the use of Molecular Beacons (see chapter 3 section 4) or short probes with much lower
Tms
[111]
and all of these, and others, are more-or-less effective, albeit not without disadvantages.
These usually are associated with more difficult probe design, additional costs for synthesis and
labeling and more stringent reaction conditions
[112]
. Optimal LNA assays can be difficult to design
because LNA nucleotides have a rather high self-affinity and a tendency to form secondary structures.
This can result in DNALNAs hybrids that are so stable that the probe is unable to hybridise with
complementary, single-stranded DNA targets
[113]
.
Hydrophobic nucleic acid (HyNA) is a less well-known, but in many ways superior alternative to
LNA and when incorporated into structured fluorescently labelled probes can be used for expression
analysis and the discrimination of alleles, SNPs, mutations and methylation status. HyNA is
composed of standard DNA nucleotides and hydrophobic stabilising intercalators (pentabases).
Pentabases are flat heteroaromatic and hydrophobic molecules, which stack on top of each when they
cannot intercalate between base pairs in a complementary duplex. This minimises the hydrophobic
surface exposure to water and hence maximises the entropy of the system. Since HyNA is mostly
composed of normal DNA nucleotides it tends to behave like DNA, without the self-affinity issues
displayed by LNA. During DNA synthesis, pentabases are incorporated inbetween nucleotides into
the backbone of the growing chain of HyNA using standard chemistries and conditions
[114]
. The
placement of the pentabases within the probe is very flexible and they can act as lids on the duplex
when placed at the ends or glue the duplex together when placed internally. Probes come in two
configuration: EasyBeacons, which are similar to Molecular Beacons and HydrolEasy probes,
which can replace MGB- or LNA-modified hydrolysis probes. When the former bind to a
complementary target, fluorochrome and the quencher are separated in spatial distance allowing the
fluorochrome to emit light. The crucial difference with beacons is that EasyBeacons do not require
an internal stem since the hydrophobic interactions of the pentabases in the EasyBeacons ensure
that quencher and fluorochrome are kept in close proximity and hence result in an efficiently quenched
probe with even lower background fluorescence than that of Molecular Beacons. The HydrolEast
probes have significantly better signal-to-noise ratios as well as higher specificity and sensitivity
because in solution the fluorochrome and the quencher are kept in closer proximity by hydrophobic
interactions between pentabases. During the amplification reaction the HydrolEasy probe degrades
in the same way as normal hydrolysis probes.
Pentabases are commercially available, and are just one of several specialist reagents that are
leading to significant improvements to the specificity and the sensitivity of the qPCR assay.
Conclusions
A successful PCR reaction requires an optimal combination of sample, primers, enzyme and reaction
buffer. Modern qPCR assays have come a long way from the use of a single heatstable DNA
polymerase and a simple buffer containing KCl and MgCl
2
, with some BSA thrown in for good
measure. Today every vendor has different polymerases with their own complex buffer of unknown
composition, targeting different applications and characterised by significantly different performance.
Their diverse properties result in different results, hence it is vital to know what enzyme was used
with a particular PCR reaction.
On the other hand, the use of specialised enzymes and buffers has resulted in more sensitive and
specific PCR assays. Together with improvements in primer and dNTP quality, this has placed a set
of basic reagents into researchers hands that holds out the promise of improved performance as well
as increased reliability of the results.
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The Polymerase Chain Reaction

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