Definitive Pcr

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he Polymerase Chain Reaction

hapter 1

roduction

e live in an age where hype and exaggeration have become so pervasive that it is difficult to finequate terms for something really extraordinary. Furthermore, impatience, haste and short attenans seem to be defining adages for our times, inviting acclaim for technological bandwagons th

efly promise the earth, but then fail to deliver because the technologies were either conceivedste without proper regard for technical and biological concerns or become superseded by the nhnological revolution.

e polymerase chain reaction (PCR) is the antithesis of such technologies and richly merits all tplification it attracts. Its conceptual clarity, practical minimalism and ubiquitous applicabilityke it the wonder technology of the molecular biology age.

ccess has many fathers, and the PCR is certainly no orphan. The truth behind what steps, whos

ntributions and which timelines were critical to the invention of the PCR will probably never bown , but the public face of PCR is Kary Mullis, who was jointly awarded the 1993 Nobel Priemistry "for contributions to the developments of methods within DNA-based chemistry" and

ecifically for "his invention of the polymerase chain reaction (PCR) method". The other half wMichael Smith for his fundamental contributions to site-directed mutagenesis. Whether the rathmantic story of its invention in Mullis book is how it really happened will forever remain a mint. Its influence on modern science, however, cannot be overemphasised and its hold on theagination is also worth recalling. Hence it is amusing, and probably telling, that the officialWe of the Nobel Prizechooses to highlight amongst the uses of the PCR method a science fiction

plication of PCR: its role in the film "Jurassic Park", where it is used to recreate extinct dinoss also funny, and really quite telling, that the site refers to them as giant reptiles.

PCR initiated a revolution, real-time PCR (qPCR) not only cemented its achievements but extem into areas inaccessible to conventional PCR. Its inventor, Russ Higuchi, deserves his placeall-time giant of science for realising not only that a fortuitous finding could have such tremenplications, but developing all the concepts and practices that are still being followed today.

CR ingredients

e PCR is a model enzymatic reaction that results in the synthesis of virtually unlimited copies ecific DNA from a mixture containing numerous different DNA molecules. A PCR reaction haslowing requirements:Knowledge of at least some of the sequence of the target DNA molecule. This does not

ve to be the exact sequence, and it is possible to amplify sequences that are somewhat dissimilA 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 DNAdepend


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NA polymerases can extend only pre-existing chains; they cannot directde novosynthesis byning two deoxyribonucleoside-5'-phosphates together to make the initial phosphodiester bondneral, PCR primers are 18-24 nucleotides in length and are specific to complementary sequencopposite strands of their target DNA. However, they can be longer, for example if they have alymerase promoter site at their 5-ends and may have some mismatches (degenerate primers), ng as these are not at the 3-end of the primer sequence.

A reaction mixture containing K+and Mg2+or Mn2+, all four deoxynucleoside triphosphates

NTPs) and a DNA-dependent DNA polymerase. dUTP is sometimes added to prevent carry-ovntamination[1]and the polymerase should be heat stable, although of course the initial experimre carried out with a thermolabile enzyme.

A thermal cycler that controls and rapidly varies the temperatures of the PCR reaction mixturermal cyclers are now programmable, but the process was initially carried out by manual tranreaction tubes from one water bath or heating block to another. Cycling involves a denaturatiop between 92C and 95C, which breaks the hydrogen bonds holding double stranded DNADNA) together, an annealing/polymerisation step usually between 50C and 65C, which allo

timal hybridisation of primers to their complementary target sequences on the DNA template all as their initial or complete extension by the polymerase and an (optional for SYBR Greenlymerisation step usually between 70C and 72C, which allows the DNA polymerase to initiad 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, endR or by real-time detection of fluorescent signals that are directly proportional to the number plification products, usually known as amplicons, generated during each amplification cycle.

CR methodology

first sight the PCR reaction is rather straightforward:is initiated by combining a DNA sample at low concentration with a forward (sense) and rev

ntisense) primer pair in a 10mM Tris-HCl, pH 8.3, 50mM KCl reaction buffer containinguimolar ratios of four deoxynucleoside triphosphates, together with Mg2+and a thermostable Dpendent polymerase, usually Taqpolymerase

ome home-brew buffers contain in addition bovine serum albumin (BSA), - mercaptoethan

d NaCl, and modified bases such as biotin-11-dUTP and 7-deaza-dGTP may also be includedpending on the aim of amplification.

his mixture is heated to around 95C for a period of time, which used to be 15 seconds but theys can be as short as one second for amplification targets up to 500 base pairs (bp). Thisnaturation step separates the complementary DNA strands, leaving them single stranded and optargeting by the primers that are present in vast excess. The trick here is to heat the sample to twest denaturation temperature for the shortest possible time, so maximising both strand separawell as enzyme stability.

he mixture is rapidly cooled to the annealing temperature, which is usually somewhere betwe


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C and 60C and is held for increasingly short times, with one second perfectly feasible these th fast reagents and SYBR Green-based chemistry. If the primers find their complementaryquences, they base pair with the template DNA, forming a short, double-stranded region.

rimers must possess a free 3'-OH end to which an incoming deoxynucleoside monophosphate ded by theTaqpolymerase. The deoxynucleoside monophosphate to be incorporated is chosenough its geometric fit with the template base to form a WatsonCrick base pair. AsTaqpolymealyses the successive addition of deoxynucleotide units to the 3'-end of the primer, primer and

mplate complex are stabilised. Many protocols, especially those using probe-based chemistriely two temperatures, as the polymerase has sufficient activity at 60C (approximately 50%) tomplete the polymerisation process and it helps ensure that the probe remains hybridised to itsmplate until displaced and cleaved by the polymerase.

n the three-step protocol used for conventional and DNA-binding dye-based chemistries, themperature is raised to 72C, close to the optimal temperature forTaqpolymerase allowing thelymerase to generate specific amplification products.

n vivoDNA synthesis is always in the 5'3' direction; hence the PCR reaction proceeds tonthesise a polynucleotide sequence that runs antiparallel and complementary to the template unch newly synthesised strand reaches the end of the complementary sequence delineated by the d of the opposite primer.

n theory, this results in a doubling of the amount of original template DNA present in the PCRution. Since the product of one cycle serves as the template for the next cycle, PCR leads to thponential amplification of the initial DNA template, producing over 1x106copies of a homoge

R product in 20 cycles[2].

nce, given a known DNA sequence, it is possible to amplify it specifically from every other Dolecule that surrounds it.

he aim is to target RNA, this is achieved by adding a preceding reverse transcription step andrforming the PCR reaction on the resulting cDNA sample. Whilst this allows the detection oflular RNAs, including their localisation usingin situRT-PCR, it also opens up the field ofgnostics to permit the sensitive and specific detection of RNA viruses.

T-qPCR will be discussed in a separate volume in this series, but it is worth mentioning that thdition of the reverse transcription step changes the nature of the qPCR assay. It requires carefuality control of the RNA templates being investigated, assessment for RT-inhibition, and theriability of the RT step can introduce significant errors and uncertainty into the quantificationcles (Cqs) recorded at the end of the PCR step. These technical difficulties are exacerbated byriable analysis methodologies, inappropriate normalisation procedures and non-transparentporting in the peer-reviewed literature.

PCR reaction has three distinct phases:

The early cycles:These require optimal primer specificity. Approximately 1014primer molec


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arch for their complementary sequences by binding transiently to random sequences, rapidlysociating if they are non-complementary and reannealing elsewhere. Specificity is determinedannealing conditions,i.e.the temperature and divalent cation concentration, that must be optim

favour the hybridisation of perfectly matched duplexes for a period sufficiently long for thelymerase to form a ternary complex and initiate DNA synthesis from the primer.

The mid cycles:These require optimal amplification efficiency. Here the increasing number ofmplementary targets results in more efficient primer scanning, thus allowing the amplification

ocess operating at maximum efficiency. Ideally, this will result in a doubling of the number of quences 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. Twhen amplification becomes suboptimal due to inhibition of the DNA polymerase, present atund 3x 1010copies, by accumulated target DNA or if not every amplicon is used as a template

cause there are more amplicons than polymerase. In addition, at high amplicon concentration tmplementary strands are more likely to find each other and start annealing at a higher temperatun the primer/template combinations and so will be removed from participation in the next cycPCR reaction.

ficiency of amplification

e PCR process is termed a chain reaction because the products from one cycle of amplificatve as the substrates for the next one. This results in a series of amplicon doubling events withcle of the PCR reaction, defined as 2n, where n equals the number of cycles. Theoretically, theponential increase in the amount of amplification product is described in equation 1 and plottegure 3A.

=N02n(1)=number of amplicons, N0is the initial number of molecules, n is the number of amplification

clesis equation denotes the linear relationship between the number of amplified target molecules ainitial number of target molecules, as shown in Figure 3B.

wever, the theoretical efficiency of amplification is not the same as the empirical efficiency aely 100%. Hence it is necessary to modify equation (1) to add an efficiency correction factor aown in equation (2)

=N0(1+E)n(2)

amplification efficiency.

e exponential nature of the PCR process means that a small change in amplification efficiency ult in significant differences in the amount of product generated, regardless of whether the numinitial target molecules was the same. For example, if reactions A and B have amplificationiciencies of 85% and 95%, respectively, after 40 cycles reaction A would generate a 4.86x10

d increase in the amount of target molecules, whereas reaction B would generate a much great


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9x1011-fold increase, which is more than eight-fold more.

e amplification efficiency is affected by several experimental factors, with primer structure,plicon structure, sequence and length as well as sample purity being critical parameters. Thes

ntribute to the observation that the yield of amplification product can differ even if the same ta

quence, cycling conditions and reagents are used[3-5]. Moreover, this variability tends to be

predictable and can be significant[6].

portantly, the amount of PCR product levels off as the rate of amplification slows, resulting in ateau effect described earlier. The number of cycles required to reach the plateau phase variesgely depends on the number of original target molecules, but may also be sequence-dependentis variability obscures the linear relationship between initial and final template copy numbersmakes conventional PCR unreliable as a quantitative technique.

CR-theory to practice

e principle of PCR was first described in 1971[7](with an incorrect apostrophe in its title) an

rth reading the visionary description of this reaction. There can be no doubt that this paperscribes the essence of the PCR and publicly, albeit theoretically, describes this technique.

, why did Kleppe et alnot pursue their revolutionary and simple concept? Why was the firs

actical demonstration of the PCR not published until 1985[8], and then by a different group? Whd it take another 14 years for this vision to be translated into reality?

member that molecular biology was in its infancy, with the first restriction enzyme (HindII)ncidentally isolated in 1970. So, what was lacking at that time were various crucial componen

nowadays take for granted, but which at the time of Kleppes thinking about his theoreticalperiment were simply not available. Amongst these there are three elements in particular that st:

A reliable, fast and cheap way of preparing oligonucleotide primers of 15 to 25 nucleotides. Imember watching a post-doc struggling to synthesise an 8-mer back in 1983, with messy chemnual operations and profusions of bad language.

A DNA polymerase that could survive the repeated rounds of heating and cooling without requ

lenishment after each cycle. Opening the caps of 50 tubes every few minutes to add fresh enzynot really a recipe for reproducible results.

Automated thermal cyclers with reliable temperature ramping and holding. No technique, no mw powerful, could thrive if it meant sitting for two hours next to a set of waterbaths and transfks of tubes every minute from one to the next.

igonucleotide primers

vances in oligonucleotide synthesis chemistries, coupled to improved purification and qualityntrol processes, have resolved the first challenge. Together, they have resulted in substantial


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reases in primer quality, yield and length, crucially combined with the all-important crash in cigonucleotides have become a commodity, purchased in bulk at rock-bottom prices and availam numerous competing oligonucleotide manufacturers.

e major advance was the substitution of the chloride leaving group present on a phosphite-trie

th the amine leaving group on a phosphoramidite monomer[9-11] (also see chapter 1, section 2)sulting nucleoside phosphoramidites are stable nucleic acid monomers with an acid-labile

methoxytrityl leaving group at their 5-end and a base-labile -cyanoethyl protected 3'-phosph

oup at its 3-end. This modification made it possible to synthesise phosphoramidites in advanclate them as stable solids and store them until required, thus enabling commercial synthesis antribution of DNA synthesis reagents.

e first monomer is attached through its 3 carbon to a glass or polystyrene bead with surface hd channels. Hence synthesis begins with the 3-most nucleotide and proceeds through a series protection, coupling, capping, and stabilisation cycles that result in sequential additions to the d of the growing oligonucleotide until the 5-most nucleotide is attached. The high couplingiciency (typically >99%) permits the manufacture of long oligonucleotides in excess of 100 ba

lid phase synthesis allows excess reagents to be washed away and avoids polymerisation thatuld occur in a solution phase reaction. The introduction of tetrazole catalysis[12]for

osphoramidite activation just prior to coupling completed the breakthrough that was essential R to become a ubiquitous technology, rather than a plaything for chemists.

ermostable DNA polymerase

e second constraint was removed by the discovery of todays most commonly used DNAlymerase, identified fromThermus aquaticus, hence its nameTaqpolymerase, a bacterium tha

es in thermal hot springs and and depends on enzymes that are resilient to inactivation by highmperatures[13, 14]. Taqpolymerases half-life is 130 minutes at 92.5C, 40 minutes at 95C and

nutes at 97.5C[14]. Based on sequence similarity toEscherichia coliDNA polymerase I,Taq

lymerase has been assigned to the A family of DNA polymerases[15]and, likeE. coliDNA Po

ssesses an intrinsic 53 exonuclease (nick translation) activity[16]. Its structure-dependentgle-stranded endonuclease activity allowsTaqpolymerase to cleave 5 terminal nucleotides ouble-stranded DNA, releasing mono- and oligonucleotides. The preferred substrate for cleavaplaced single-stranded DNA, which assumes a fork-like structure; hydrolysis occurs at the

osphodiester bond joining the displaced single-stranded region with the base-paired portion oand.Taqpolymerase has no 35 exonuclease (proofreading) activity[14, 17]. Its introductionR had several important consequences:

CR could be automated, since there was no longer any need to add DNA polymerase after eacnaturation step.

ubes no longer needed to be opened after the completion of every PCR cycle. This reduced thcontamination by PCR amplification products, although of course the tubes still needed to be

ened after the PCR reaction was completed. The resulting aerosols certainly contributed to low


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el contamination that was present in every PCR laboratory.

inceTaqpolymerase has an optimum reactivity (Vmax) between 70C and 80C and significaidual activity between 55C and 70C, the polymerisation step can be performed at 72C, rathn at the original 37C. This results in reduced secondary structures, speedy stabilisation andension of annealed primers concomitant with a huge improvement in replication specificity. Tde recourse to a Southern blot less obligatory, since it allowed the viewing of (more or less)gle bands on ethidium bromide stained agarose gels.

he increased processivity ofTaqpolymerase, 50-80 nucleotides/second at 60C before

sociating from the DNA template[18], compared with the 20-40 nucleotides/second of Klenowultes in the amplification of significantly longer fragments (4,000 bp up to 10,000 bp) than wassible with Klenow (400 bp).

he absence of a 3-5 (proofreading) exonuclease activity makesTaqpolymerase faster thanenow.

Taqpolymerase generates significantly higher yields of PCR amplicon than Klenow [19].

Taqpolymerase is less sensitive to inhibition than Klenow, making for more robust PCR protohough there is some suggestion that it is sensitive to proteolytic degradation.ermal cyclers

ermal cyclers are programmable cycling incubators that automatically and precisely regulate aange temperatures for DNA denaturation, primer annealing, and primer elongation at definedervals. They usually incorporate a thermal block that holds individual tubes, strips of tubes orcrotitre plates. Rapid heat transfer from the heating block to the in-tube sample liquid ensures

gh efficiency of amplification and a thermal processor must enable temperature uniformity for a

mples within an individual run as well as run-to-run repeatability. The inadequacy of early heaocks led to the development of water bath as well as rotor-based thermal cyclers andnotechnology is beginning to have an impact on the latest thermal cycler designs that incorporacrofluidic chips with pico- or nanolitre volumes.

tus Instrument Systems developed the first thermal cycler, an aluminium block that could be hed cooled as required and in a joint venture with PerkinElmer introduced the first fully automatR unit in the 1980s (Mr. Cycle). Why Mr. Cycle? It is not obvious to a nonAmerican, but Ruguchi informed me that it was named after a coffee machine popular in the USA, although its na

s unfortunate if it ever missed a cycle. Another early instrument was a prototype called Baue (because of its beautiful blue colour) and was devised in 1986 to study HIV. It was the firsodel that combined the software controlling the process with the heating and cooling block in ochine. It is on show at the Science Museum in London and more information isavailable onlin

ccessful amplification of a DNA target depends on several variables:Actual temperatures inside the sampleUniformity of heating and cooling across the blockRamp times of the thermal cycler

Degree of convection in the sample


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Design of the plasticware holding the samples

rly designs had significant issues with temperature homogeneity[20]and accuracy[21], variable

rformance[22]; even more recent designs do not always perform within the manufacturers

ecification[23]and performance continues to be limited by spatial variation across the block[24

vertheless, in general todays instruments are not just robust, accurate and capable of very higoughput, but are also (relatively) inexpensive and easy to use.

fferent sample volumes may require an adjustment to the incubation times to maximise thermaluilibration of the reagents. One of the banes of PCR-of-old was the need to use oil to seal in thction to keep it from condensing inside the lid of the tube. This removes water from the reactixture and so concentrates the salts and other reaction components. Luckily, modern thermal cyve heated lids, which maintain a constant temperature of around 105C. This ensures that themperature of the exposed portions of the tubes or wells is raised and keeps condensation to animum. Ramp time, which refers to the time it takes the heated block to change from one

mperature to another, is usually longer than either denaturation or primer annealing times and thorter it is the better.

al-time PCR

w did Russ Higuchi invent qPCR? As he himself recalls, he had been working on a project thavolved the use of biotinylated oligonucleotide primers and streptavidin to determine whether Puld be used to generate long branched chains of amplicons. Unfortunately all that appeared toppen was that DNA precipitates formed, which were visualised by the manual addition of EtBer the PCR step, followed by UV illumination. At one stage Russs technician, Bob Griffith,came fed up with having to add EtBr each time after the PCR and added it to the mastermix

forehand. On one occasion, whilst looking at a band on a gel, he mentioned to Russ that thatrticular PCR reaction had proceeded with EtBr in the reaction, something that should have notssible since EtBr is a known inhibitor of DNA polymerases. They immediately did the experimain, this time with a no template control (NTC), which did not show any sign of specificplification. When they repeated the PCR without the streptavidin and illuminated the reaction th a UV light, the tube containing target DNA lit up brightly, whereas the NTC did not. Of coury got very excited, since this suggested that, given the right conditions, addition of EtBr to the ction might allow the detection of the amplified DNA through increased fluorescence without

ed to open the reaction tube. Furthermore, it immediately occurred to Russ that a continuousonitoring of the PCR reaction, rather than endpoint detection, would be a useful feature of this nthodology.

he hooked up a thermal cycler to a spectrofluorometer and found that he could indeed follow tR reaction in real time. The output was very simple, with the fluorescence trace at 600nmording peaks and troughs corresponding to readings at 50C and 94C, respectively. In additiore was a net increase in fluorescence at the lower temperature after each cycle, correspondingincreasing amount of amplicon made during each polymerisation step. Together with Bob Wa

ss placed a charge-coupled device (CCD) camera so that it looked directly down at the reacties sitting in a thermal block in a darkroom and illuminated the setup with a UV light. An image


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s taken at every cycle of the PCR reaction and the resulting pixel values were combined for edividual tube. This resulted in the now familiar amplification plots, but because of the differentseline fluorescence values could not be used for quantification. This is where Russs secondainwave came in. He realised that if one made the reasonable assumption that baseline fluoress the same for all samples, one could normalise PCR results relative to each other based on thly cycle fluorescence readings. This generated perfectly overlapping curves, with variationtween Cqs being fewer than 0.2 Cqs.e third brain wave was the realisation that all PCR reactions have fixed start and stop cycles,

owing amplification products to catch up with each other. Russ realised that this could be the a quantitative assay, with perfect quantification conditions right up to the plateau phase. Hevised the concept of a fluorescence threshold value and demonstrated that the number of cyclesok to cross this threshold is inversely and linearly related to the logarithm of the initial numberget molecules. These cycle numbers are now known as quantification cycles (Cq) and can beerpolated to fractions of cycles. If Cqs are compared with the Cqs obtained from a standard cuth known initial target copy numbers or amounts, the starting target number in each unknowninferred. He also realised that the slope of the standard curve was related to the per-cycleiciency of PCR replication. Since 100% efficiency equates to a perfect doubling per cycle, a

ofold dilution of starting template would result in a one-cycle difference between Cqs. If theiciency were less than 100%, the difference would be more than 1 Cq. Consequently, the slopcalibration curve describes the number of cycles required to make up for the dilution. He

scribed per-cycle efficiency as:

0-1/slope-1)x100%

is calculation showing the relationship between slope and PCR efficiency for 10-fold dilutionl in use today.esent day chemistries

esent-day qPCR assays utilise three general approaches:Non-specific DNA-binding dyes that are a further development of the first EtBr-based qPCRNon-destructive hybridisation-based assaysCombined hybridisation/hydrolysis-based assays

gardless of chemistry, the amount of fluorescence emitted is directly proportional to the numbR amplicons being synthesised, although the kinetics of fluorescent reporter increases dependtype of chemistry used. Increases (or decreases) can be either cumulative, as with hydrolysis

porters or non-cumulative, as with DNA-binding dyes or hybridisation reporters[25].

NA-binding dyes

e simplest, cheapest and most widely used approach makes use of the affinity of certain fluorees, for example SYBR Green I, for double-stranded (ds) DNA. When in solution and unboun

NA, their fluorescence emission is very low when subjected to light of an appropriate wavelene accumulation of amplicon during each PCR cycle results in the binding of increasing numbere molecules to ds DNA. This induces a conformational change that leads to hugely increasedorescence of the excited dye. Hence the effect is like turning up a dimmer switch, where with ist of the knob there is a little more light (Figure 1A). As with conventional PCR, the specifici


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reaction is determined entirely by the primers; hence the principal disadvantage of using thisthod is that both specific and non-specific products generate fluorescent signals. Its mainvantages are that there is no need for additional, expensive oligonucleotide probes and that it issible to carry out post-PCR melt point analyses to check for the presence of non-specific reacoducts.

n-destructive hybridisation-based assays

though non-destructive hybridisation assays include a wide range of different chemistries, in

actice only two or three really matter.luorescently labelled primers become incorporated into ds amplicons and fluorescence is eithreased or decreased. These primers can be simply labelled with a fluorochrome (Figure 1B),ke use of specific pairing by a synthetic bases (Figure 1C), or can be more complex and inclu

obe component at their 5-end, which in a unimolecular reaction binds to and reports the presea specific amplicons (Figure 1D).

here are several variants on the theme of a single fluorochrome/single probe that is compleme

specific targets. Following hybridisation to newly synthesised amplicons, dequenching of theporter results in the emission of fluorescence, which is detected either directly (Figure 1E) or nsferred to a DNA-binding dye via resonance energy transfer and detected as emission at its lovelength (Figure 1F).

A single oligonucleotide probe with donor and acceptor moieties attached to its 5- and 3endsngle oligonucleotides behaves like random coils in solution, the ends will come together from time, resulting in quenching by fluorescence energy transfer. Binding of the probe to its target nto a linear conformation, prevents interaction between donor and acceptor and results in

orescence emission (Figure 1G).he hybridisation of a pair of non-complementary oligonucleotide probes labelled at theirpective 5 and 3 ends to adjacent sites on a target strand brings a donor and acceptor moiety each other, resulting in resonance energy transfer and detection of fluorescence emission from

ceptor moiety at a different wavelength[11]. (Figure 1H).

wo complementary oligonucleotides, one of which contains a donor fluorochrome at its 5-enher contains an acceptor moiety at its 3end (chapter 3 section 3), are annealed and emit no

orescence in the absence of complementary targets. Since small complementary oligonucleotidplace and bind to each other in a dynamic equilibrium, PCR amplicons compete for binding toobes during the PCR reaction. This separates the labelled oligonucleotides and results inorescence emission (Figure 7K).

he probe sequence can contain additional complementary sequences at either end that anneal isence of target to generate a hairpin stem and so bring the terminal fluorochrome/quencher moo close contact. In the presence of target complementary to the probe sequence, a relatively rigobetarget hybrid is created, which disrupts the stem structure and separates donor from accep

ulting in fluorescence (Figure 1L).


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mbined hybridisation/hydrolysis-based assays

bridisation/hydrolysis-based chemistries are variations on the basic theme of attaching fluoresnor and acceptor moieties to the same oligonucleotide, which can be either a targetspecific or versal probe. These take on a random coil conformation in solution and fluorescence is quenc

n the most popular embodiment, presence of target results in the dual-labelled probe binding toplicon, followed by cleavage by the 53 activity ofTaqDNA polymerase. This results in t

paration of fluorochrome and quencher with concomitant emission of fluorescence (Figure 1MA second hydrolysis-based approach uses a primer with an antisense DNAzyme binding/ cleave at its 5-end that becomes activated during the PCR reaction and cleaves a fluorescently labeiversal probe (Figure 1N).

Alternatively, the target-specific probe may be separated into two sections that together specifyNAzyme recognition and cleavage site, so that the universal probe is hydrolysed only in theesence of a complementary target that is bound by both sections that binds to the universal probd cleaves it (Figure 1O).

thin each class, there are numerous variants, most of which will be described in more detail inevant chapters of this book. They provide an immense flexibility for assay design, allowing a oice of chemistries to suit each task on hand.

ure 1. (overleaf) A.DNA dye-based chemistry. The transformation from free dye in solution (blue) to dsDNA binding (glowingen) changes the conformation of the dye and results in fluorescence emission (SYBR Green I). B. The hairpin structure of th

mer with the fluorochrome attached towards the 3-end of the molecule (green) opens up during the PCR, dequenching therochrome (glowing green) (Lux).C.The 5-end of the primer contains a iso-dC (red base) covalently linked to a fluorochromabsence of a target, the fluorochrome emits fluorescence (glowing green). In the presence of target, the iso-dG, which is covaed to a quencher, becomes incorporated opposite the iso-dC and quenches its fluorescence (Plexor).D.The 5-end of the pra blocker(red diamond) and a quencher (black) and fluorochrome (green) linked by a target-specific probe sequence and a stecture. Priming from the 3-end results in an amplification product which on cooling is targeted by the probe sequence in amolecular reaction (glowing green)(Scorpions).E.A single fluorochrome (green) is quenched by its surrounding ss DNA seqch is also blocked at its 3-end to prevent extension (red diamons). Upon hybridisation, the fluorochrome is dequenched (glowinen).(e.g. Hybeacons).F. In addition to the single labelled probe, DNA binding-dyes (blue) are added. During the annealing stbe and dyes bind to target template and FRET from one to the other (glowing green) can be detected (Resonsense).G.A duen/black) on a ss probe will be quenched due to the oligonucleotide assuming a random coil formation. Upon hybridisation,rochrome and quencher are separated and fluorescence is emitted (glowing green).H.A single fluorescent label is at the 5- 3- (red) ends, respectively, of two probes that target adjacent sequences. Following hybridisation, fluorescence emission due t

ET is detected (Lightcycler probes).K.Two complementary oligonucleotides form a doublestranded structure, with one labeh a fluorochrome at its 5-end and the other with a quencher at its 3-end. Following a denaturation step, the two strands of theonucleotide duplex are separated. Upon annealing in the presence of target, the quencher-labelled strand is displaced by the tarthe fluorochrome emits fluorescence. L. A probe with terminal complementary sequences forms a hairpin structure withrochrome (green) and quencher (black) physically close together. Upon hybridisation, the arms open and fluorescence is emitt

olecular Beacons). M.A dual labelled probe binds to its target and is hydrolysed by the nuclease activity of Taq polymeraseqMan ). N.The 5end of the primer contains an antisense DNAzyme binding and active site (blue/red/blue). Upon replicative site is created and cleaves the universal probe at its cleavage site (blue diamond) (Qzyme).O.Same as N, except that theAzyme is made up of two components (MNAzyme).


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Figure 1. Selecti

CR chemistries.otein-targeted PCR

e power of PCR technology has been extended to permit the detection of proteins.muno-PCR

e of the main questions arising from PCR-, especially RT-PCR based results is how any nucled quantification relates antibodies to detect the to protein expression. Traditionally, this hasquired the use of relevant proteins using western blots, immunohistochemistry or

munoassays. Immunoassays such as the enzyme-linked immunosorbent assay (ELISA) have lonen the mainstay of protein quantification and are widely used in microbiological diagnostics, wir power lies in their ability to identify pathogens directly by detecting pathogen-specific protwell indirectly by detecting antibodies produced against them. However, despite its specificitye of ELISAs can be limited by their lack of sensitivity. Hence the idea of combining the advant

ELISAs and the PCR to create a powerful and versatile method for the detection of low quantiprotein antigens as well the antibodies that are generated against those antigens. That techniqu

led immuno-PCR (iPCR) and has been around for 20 years[26](Figure 2).

CR represents an inversion of conventional ELISA protocols: whereas ELISA uses antibodyennjugates with the enzymes substrate added subsequently as a freely diffusing species, in iPCRbstrate (the DNA template), is linked to the antibody while the enzyme is added subsequently.mplification of the DNA marker through PCR enhances the limit-of detection (LOD) of a given

ISA by between 100-10 000-fold. Nevertheless, its main drawback is the problem of cross-

ctivity and nonspecific adsorption, which sets the limit for its selectivity. Hence the practicalplication of iPCR has been somewhat stifled by complex protocols and problems of backgroun


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ise. For example, physically linking antibodies with DNA turns out to be quite tricky, since the

streptavidin protein A chimera[26]limits its application to direct detection only, whereas the u

avidin[27]leads to the formation of different species of conjugates and results in high backgrouise causing reduced sensitivity and reproducibility. However, the last few years have seen majvances in the development of new linker molecules, new formats, the association of iPCR withnotechnology systems and the availability of ready-to-use reagents from commercial providerss laborious protocols.

gure 2. Basic principles of ELISA and iPCRA.In the ELISA a signal is generated by the act

an enzyme linked to a detection antibody.B. In iPCR the signal is generated by a qPCRaction primed by the DNA linked to the detection antibody. In practice, the detection antibo

otinylated and liked through streptavidin to a biotinylated DNA.

e method has also been simplified and a universal iPCR protocol based on the in situassemblotinylated DNA, streptavidin and biotinylated antibody-antigen complexes has become the mos

dely used format for research applications[28]. It can be used in both direct and sandwich formquires less hands-on time with far fewer washes needed to eliminate carryover contamination aplays much reduced nonspecific binding. As a result of these and other improvements iPCR h

ned in robustness and by linking up with other methods such as bead technologies or phage dibeginning to find a broad variety of applications [29]. Standardisation is likely to be enhanced be of commercial, tailored reagents and kits solutions for specific analytical tasks. Since DNA, d protein detection can be carried out using the same qPCR instrument, it is obvious that iPCRays can be used to link information on genetic status, miRNA and mRNA expression and prote

vels.

CR may be of most use when looking for very low abundant targets in quality control, diagnostd analytics applications. For example, multiplex iPCR may be useful for ultrasensitive detectiod quantification of tiny amounts of target antigens when monitoring complex medical response


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tterns of individual patients following treatment. Another area which also calls for ultrasensitialyses concerns the monitoring of biological compounds such as contaminants in food, either bxins or genetically altered protein components. It will also be interesting to see its application

field of single cell analysis, with early indication suggesting that this technology may have a r

play[30]. On the other hand, technologies come and go and the proximity ligation assay (PLA) mll prove to be more robust, sensitive and reliable than iPCR.

oximity ligation assay (PLA)

though PLA is similar to iPCR, the big difference between the two is that PLA results depend binding of two, three or more antibodies to a specific target protein. In practice, the antibodie

ve been conjugated to different oligonucleotides to form proximity probes. The antibodiesognise two or more different epitopes on the same specific target protein or can be used to de

otein complexes. When antibodies concurrently bind to their targets, the oligonucleotides carriproximity probes are physically brought together (into proximity); a connector oligonucleo

bridises to the oligonucleotides and acts as a template for their ligation into a full-length molecgure 3). This results in a chimeric DNA strand that can be amplified and detected by qPCR or

her detection methods, making PLA several orders of magnitude more sensitive than westernots[31; 32]. A further development is its use for the detection of ternary complexes, where three

oximity probes give rise to the amplifiable DNA molecule[33]. Most recently the assay has beeodified to allow the detection of small molecules, which had been difficult since their small

olecular structure prevents two antibodies from binding[34]. The new assay, termed competitivmunomagnetic-proximity ligation assay (CIPLA) uses a single antibody to target clenbuterol antopamine competitively, with LODs 10-50-fold lower than ELISAs.

viously assay performance is closely linked to the affinity and specificity of the antibody, sinc

bsaturating amounts of antibody are used to minimise background noise[35]. The requirement foo independent binding events reduces the likelihood of them occurring in the absence of theecific target protein, and further minimises the background signal from nonspecific or crossreaibody binding, so contributing significantly to the high specificity and sensitivity of PLA

hnology [31; 32; 36].


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gure 3. Proximity ligation assay scheme. A The target protein is bound by two proximity pro

d the oligonucleotides are brought in proximity. B. A connector oligonucleotide can hybrid

th oligonucleotides, creating a template for ligation. C. An antisense primer synthesises a

mplementary strand.D. The newly created DNA-molecule can now be amplified by qPCR.

A was first described in 2002, and used a DNA aptamer as the protein binding affinity agent[3

wever, the number of available aptamers is limited, hence antibodies are used that have beennctionalised by either direct covalent coupling of an oligonucleotide or noncovalently by incub

otinylated antibodies with a streptavidin-modified oligonucleotide[36]. PLAs can be carried ouveral formats:

s homogeneous, including multiplex[38], assays where binding, ligation, and amplification occuution, as in qPCR

n a solid-phase format where the target protein is first immobilised on a solid-phase support u

apture antibody, then detected with a PLA for the captured protein[39-42]

n situwhere the connector oligonucleotide generates a circular DNA strand upon hybridisationpaired proximity probes. Following ligation, rolling circle amplification of the circular temphich remains hybridised to the proximity probes, by phi29 DNA polymerase results in localise

plification of the ligation product[43; 44]. This technology has been commercialised asDuolinkink Bioscience.

e word of caution: a recent publication shows that various non-linear effects in the in situPLAction make it a semiquantitative measure of protein co-localisation and suggests that caution

ould be exercised when interpreting PLA data in a quantitative way[45]. Nonetheless, the poten

this method is huge with obvious applications in diagnostics, where it has been shown to impr


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accuracy of pancreatic cancer diagnosis[46]and in personalised medicine in general[47].

fe Technologies have commercialised a hydrolysis-probe-based PLA assaythat expresses resuterms of the familiar Cq. However, whilst Cq levels obtained from RNA targets are normaliseainst validated internal reference genes to account for sampling variation, there are currently ntable endogenous controls available for qPCR-based LPAs. Hence Cqs are normalised to totaunt or total protein concentration. A plot of Cq values against total cell count results in a sigmorve, in contrast to the straight-line plots typically derived from a nucleic acid dilution series. T

because Cq values are not just a result of the qPCR component, but are also influenced by probnding and ligation events. Furthermore, because the slope of the linear range of hydrolysis-prosed PLA assays depends on multiple kinetic components, the slope of a dilution series may vam sample to sample.

nclusion

the 30-odd years since the first paper demonstrating its practical use, PCR has become theolecular enabling technologypar excellence. It has revolutionised all areas of the life sciences

dicine, veterinary and agricultural sciences, forensics and many other small niche areas, makimost widely used molecular technology today.

wever, conventional PCR has several disadvantages: it is an endpoint assay, i.e.target detectcurs as a separate step after the enzymatic reaction has been completed, involving analysis ofplification products from the plateau phase of the PCR reaction, where the PCR product is no

nger being doubled at each cycle. Consequently, PCR gel electrophoresis shows broadly similount of product DNA independent of the initial amount of template. That plateau will differ fo

ch assay due to the different reaction kinetics for each sample; hence this stage is highly

onsistent between samples and is an important contributor to the frequent lack of reproducibild accuracy of conventional PCR data. Conventional PCR is also labour-intensive, subject tontamination and not easily automated or adapted for high throughput applications. Furthermoreults are qualitative, and the acquisition of quantitative data using end-point PCR requires theablishment of additional empirical quantification parameters, e.g. competitive PCR, that vary ch assay, are tedious to reproduce and are not a trivial matter.

is limitation has resulted in the development of real-time PCR (qPCR), a technology that contirevolutionise molecular biology by making it possible to quantify minute quantities of DNA an

NA with extraordinary speed and precision in a broad range of samples. qPCR can be combineth a reverse transcription step to quantify RNA or with antibodies to quantify proteins or protemplexes.

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hapter 2


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ntroduction

hen asked casually: Who discovered DNA?, most people considering themselves scientificerate would probably say Watson and Crick and perhaps also mention Rosalind Franklin. Thght even point at the famous Nature publication of 1953. What a surprise then that the correctswer is Miescher and the date was 1869.

hann Friedrich Miescher, a Swiss biochemist, was the first person to isolate a substance that cprecipitated by acidifying extracted nuclei and re-dissolved when alkaline solutions were add

869). These precipitates could not be dissolved either in water, acetic acid, very dilutedrochloric acid, or in solutions of sodium chloride, and which thus could not belong to any

e hitherto known proteins. Since he was certain the substance was derived from the nuclei, hmed it nuclein (1871). He also thought that the presence of nuclein constituted an importantference between the nucleus and the cytoplasm and suggested that the nucleus function waspendent on and therefore should be defined by the presence of nuclein. By the way, the first edthe book containing Mieschers article ber the chemische Zusammensetzung der Eiterzellen

oncerning the chemical composition of pus cells) was sold in September 2011 for 7,131.eschers continued work on nuclein led him to conclude that it was a molecule with a high

olecular weight (1872) and to show that it contained carbon, nitrogen and hydrogen andosphorous, but no sulphur (1872). He affirmed that it was a multibasic acid (1872),at leastee basic acid(1874) and thenat least a four basic acid (1874). However, it was difficult torify nuclein away from proteins and so most researchers regarded nuclein as related to proteinen when Richard Altman purified the substance free from proteins, he thought he had identifiedvel subcomponent of nuclein, which he called nucleic acid, since it behaved like an acid (1889escher, on the other hand, was convinced that the two substances were the same. In 1881 Edua

charias showed that nuclein was an intergal part of the chromosome, thus combining for the firme the histological concept of chromatin with the chemical substance nuclein.

e four constituent bases, together with the sugar, phosphoric acid and, incidentally, histones, wntified and named in 1900 by Albrecht Kossel, an achievement that won him the Nobel Prize f

edicine/Physiology in 1910. In his acceptance speech he noted that the structure and functioncell nucleus]must be associated with the general processes of life.

vertheless, the importance of nuclein as a carrier of genetic information was not appreciated f

other fifty years, since it was thought impossible that the complexity of genetic information coured by a molecule made up of four bases. It seemed far more likely that only proteins, made upor more amino acids, possessed sufficient complexity for this role. The link with the earlier wMiescher and others was made in 1944 with the publication of a paper entitled Studies on themical Nature of the Substance Inducing Transformation of Pneumococcal Types: Induction ofansformation by a Deoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III" byery, MacLeod and McCarthy, which demonstrated that nuclein, now renamed as DNA was theely hereditary substance in bacteria. The role of DNA was corroborated in 1952 with theblication of a paper by Hershey and Chase Independent functions of viral protein and nucleic

growth of bacteriophage. In it they cautiously state that: protein probably has no function in t


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owth of intracellular phage. The DNA has some function.

spite the lack of knowledge concerning the biological role of nucleic acid, the importance of ironucleotide synthesis for Chemistry was appreciated from the very beginning, when Emil Fieived his 1902 Nobel Prize for the first chemical synthesis of a purine base. Nevertheless, it t

other 50 years (until 1955) for Michelson and Todd to synthesise a dithymidine dinucleotide bndensing 3-O-acetylthymidine with thymidine 3-(benzyl phosphorochloridate) 5-(di- benzyl

osphate) and subsequently removing the protecting groups1].

identally, since the synthetic material behaved towards enzymes exactly as the dinucleotidicgments obtained by degrading deoxyribonucleic acids, this achievement confirmed the thenstulate of a 3-5 linkage in DNA. The process was cumbersome and slow, with unstableermediates, but it provided the launchpad for todays vast oligonucleotide synthesis industry thn produce large amounts and very pure oligonucleotides as long as 200 bases and even beyondey can be produced with a range of modifications that further enhance their usefulness, includinincorporation of fluorescent dyes and quenchers and specialised nucleotide analogues such as

NA, that can be incorporated into standard oligonucleotide synthesis using LNA

osphoramidite monomers.

osphodiester to phosphoramidite

igonucleotide synthesis depends on protecting reactive parts of the nucleoside molecules until lymerisation reaction is started, at which point the protective element is removed and the reacn proceed. Carefully controlled reactive elements are continuously cycled from protected toprotected, resulting in the linear, step-wise production of an oligonucleotide molecule with animum of undesired reaction intermediates and products. The first complete chemical synthesi

ne was described in the early 1970, used the phosphodiester method and resulted in the 77cleotide yeast alanine transfer RNA,[2-4]. The main disadvantage of this method was the largembers of unwanted side chains on each molecule that had to be removed by time-consumingrification steps. This problem was partly solved by the introduction of two improved synthesisocedures:he phosphotriester method, which was characterised by fewer side-chain reactions,

reased stability of reaction intermediates and quicker reaction steps

he phosphate triester chemistry that phosphochlorodites, which improved the synthesis[5].

roduced more reactive nucleoside rate and efficiency of oligonucleotide

e final step towards efficient oligonucleotide synthesis was the replacement of a chloride groum the phosphochlorodite with an amine group to generate phosphoramidites, which are still b

ed today[6-8].

lid supports


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odern oligonucleotide syntheses are carried out using automated solid-phase methods carried oolid support (resin) held between filters, in columns that enable all reagents and solvents to paough freely. Resins are insoluble particles, usually controlled pore glass (CPG) or macroporolystyrene (MPPS) and assembled oligonucleotides remain covalently attached to the solid supterial via their 3'-terminal hydroxy group.

ntrolled-pore glass is rigid, non-swelling and has deep pores in which oligonucleotide synthees place. The length of oligonucleotide being synthesised determines the pore size, with 50 nm

res used for short oligonucleotides up to about 40 bases in length. Since the growinggonucleotide blocks the pores and reduces diffusion of the reagents through the matrix, longergonucleotides require larger pores, with 100nm pores used for the synthesis of oligonucleotid100 bases in length, and 200 or even 300nm pores for longer ones.

PPS used for oligonucleotide synthesis is a highly cross-linked, low-swelling polystyrene obtapolymerisation of divinylbenzene, styrene, and 4-chloromethylstyrene in the presence of arogeneous agent. The main advantage of highly cross-linked polystyrene beads is that theyiciently exclude moisture and allow very efficient oligonucleotide synthesis, particularly on sm

ale.

lid-phase synthesis has several advantages over solution synthesis:eactions can be quickly driven to completion by using large excesses of solution-phase reagenince impurities and excess reagents are washed away, no purification is required after each stemove unwanted residual reagentshe process is easily automated on computer-controlled solid-phase synthesisers.osphoramidite synthesis

oxyribonucleoside phosphoramidite synthesis comprises four main stages that are repeatedclically to add each new specific nucleoside to the growing oligonucleotide chain. Synthesisoceeds in the 3- to 5-direction, with one nucleotide added per synthesis cycle, which consists

four steps shown in Figure 4.

ep 1: De-blocking (detritylation)

the start of the oligonucleotide synthesis the first deoxyribonucleoside, A, G, C or T dependennucleoside at the 3-end of the desired oligonucleotide, is pre-attached to the resin. It is prote

ts 5-hydroxyl position with a 4,4-dimethoxytrity group which must be removed before a secose can be added. The exocyclic amines of the bases also have protecting groups attached and tosphorous atom is protected with beta-cyanoethyl and diisopropylamine.

ter a wash with acetonitrile to remove all traces of acid and reduce adventitious water, the DMoup is removed with a solution of an acid, such as 2% trichloroacetic acid (TCA) or 3%holoracetic (DCA), in an inert solvent e.g. dichloromethane or toluene. This results in themation of an orange-coloured DMT cation that absorbs in the visible region at 495 nm and isshed away; its yield is measured colourimetrically to help monitor the stepwise coupling

iciencies of the synthesis reaction. The solid support-bound oligonucleotide precursor now bee 5'-terminal hydroxyl group, which is the only reactive nucleophile on the base monomer and


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sures that the next base can only react with that site.

ep 2: Base Condensation

nce deoxyribonucleoside phosphoramidites are fairly stable and become reactive only uponotonation, the next base monomer cannot be added until it has been activated. This is achievedding excess acidic azole catalyst, e.g. tetrazole, to the deoxyribonucleoside phosphoramidite. Iotonates the diisopropylamino group of the phosphoramidite, converting it to a good leaving gr

e pKa of this acid is sufficiently high so that it does not remove the DMT group from the reageit is still sufficiently acidic to activate the phosphoramidite. The activated phosphoramidite isded in 1.5 - 20-fold excess over the support-bound material to the synthesis reaction. Theotonated leaving group is rapidly displaced by attack of the 5-hydroxyl group of the support-bcleoside, and a new phosphorus-oxygen bond is formed, creating a supportbound phosphite

ester[9]. This occurs very rapidly (20 sec onds) and efficiently (>99%). This reaction is highlynsitive to the presence of water and is commonly carried out in anhydrous acetonitrile, a goodvent for nucleophilic displacement reactions. The excess is lower with larger scale syntheses

hich also use higher concentrations of phosphoramidites. Extra tetrazole, unbound base and by-

oducts are washed away from the reaction column.

Figure 4. The

osphoramidite oligodeoxynucleotide synthesis cycle

ep 3: Capping

ficiency of coupling is a critical parameter, since the cumulative effect of a series of pooruplings results in a poor overall yield of the desired oligonucleotide and in a product that is ra

ficult to purify. This is easily envisaged if one assumes an efficiency of 50% per synthesis cycllowing cycle one, only half the oligonucleotides have added the second base. Following cycl


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o, only 25% of oligonucleotides would have all three bases, following cycle three only 12.5%gonucleotides would be complete and very soon there would be virtually no full lengthproducesent. Small differences in theoretical efficiency (Y = (E)n-1where (E) is average couplingiciency and n is the number of bases in the oligonucleotide) have a significant effect on final yoligonucleotide.en the most efficient chemistry, the most sophisticated instrumentation and the purest reagentsnnot achieve a 100% coupling efficiency; instead anything above 98% is readily achievable anerage stepwise yields above 99% can be attained, provided reagents are pure and anhydrous.

vertheless, coupling efficiency varies for each base both by type and position in the growinggonucleotide, with the frequency of truncated nucleotides at the 3'-end much higher than at the

d[10]. As a consequence, after every cycle of activation and coupling there will be around 1-2%reacted 5-hydroxyl groups on the resin-bound nucleotide chain. These need to be inactivated sy would otherwise take part in subsequent coupling steps, generating a series of deletions in

dition to full length oligonucleotide[11]. This is minimised by carrying out a capping step after upling reaction that blocks the unreacted 5-hydroxyl groups. An electrophilic mixture of acetichydride and N-methylimidazole (NMI), dissolved in tetrahydrofuran with the addition of a smaantity of pyridine, rapidly acetylates the 5-hydroxyl groups, rendering them inert to subsequentctions. The pyridine maintains a basic pH that prevents detritylation of the nucleosideosphoramidite by the acetic acid formed by reaction of acetic anhydride with NMI. Cappedgonucleotides remain as short species that are easily removed by a variety of purification metht all unreacted molecules are capped and continue to participate in subsequent cycles of synthulting in near full-length molecules that contain internal deletions, the so-called (n-1)mer specthough these molecules will usually work for PCR purposes, they can cause problems withecificity and should be removed by PAGE or HPLC if primers will be used in multiplex reactipping also removes any products that may have arisen from reactions of activated

osphoramidites with the O6 modification of guanosine. These can undergo depurination duringbsequent oxidation step, with any apurinic sites readily cleaved during the final deprotection ogonucleotide, resulting in shorter oligonucleotides and reducing the yield of full-length productra acetic anhydride or N-methylimidazole are removed from the column by washing.

ep 4: Oxidation

e newly formed trivalent phosphite triester linkage formed in the coupling step is acid unstableust be converted to a more stable species before the next synthesis cycle. This is achieved by ioidation in the presence of water and a weak base such as pyridine. This forms andinephosphorous adduct that is hydrolysed to yield pentavalent phosphate triester, essentially armal DNA backbone protected with a 2-cyanoethyl group, which blocks undesirable reactionsosphorus during subsequent synthesis cycles. This oxidation step usually completes one cycle gonucleotide synthesis, although some DNA synthesisers include a second capping step after

dine oxidation to dry the resin, since any residual water from the oxidation mixture can inhibit xt coupling reaction. The excess water reacts with the acylating agent to form acetic acid, whicshed away

st Synthesis


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e 3 -end of the oligonucleotide is attached to the solid support by succinyl linker, which isaffected by all the reagents used in the solid-phase oligonucleotide assembly, but is cleavable d of the synthesis. Cleavage is by treatment with concentrated ammonium hydroxide at 55C fours, which also deprotects the phosphorous by -elimination of the cyanoethyl group, and remoacetyl capping groups and the base protecting groups. The resulting aqueous solution, contain

ude mixture of product oligomer, truncated failure sequences with free 5-hydroxy ends, byprodeprotection and silicates from hydrolysis of the glass support, with more impurities accumulaoligonucleotide length increases.

this point, oligonucleotides are usually desalted, a misnomer since no salt is used duringgonucleotide synthesis. Instead desalting is a process that removes organic impurities such asnzamide and acrylonitrile and small molecule impurities such as protecting groups and shortncation products using gel filtration or organic phase extraction methods. Use of suchgonucleotides without further purification is cheap and is acceptable for the short primers usedutine PCR assays. However, truncation species can interfere with multiplex reactions or whenplicon-length oligonucleotides are needed. Hence, it is important to be aware that a desaltedgonucleotide includes a significant amount of unwanted material and that if optimal performan

quired, additional purification methods should be used. Typically, polyacrylamide gelctrophoresis (PAGE) is used to separate the oligonucleotides by size, such that only thosentaining the correct number of nucleotides are selected for further use. High performance liquiromatography (HPLC) is useful when oligonucleotides contain modified bases, as it separatesgonucleotides based on charge and/or hydrophobicity. After oligonucleotides have been purifprudent to characterise their quality, especially when synthesizing dual labelled probes or veryng oligonucleotides. This is most easily done by obtaining the molecular mass of thegonucleotide by recording its mass spectrum. This can be done either by electrospray massectrometry (ES MS) or by matrix-assisted laser desorption/ionisation time-of-flight mass

ectrometry (MALDI-TOF).

nclusions

spite the basic principles being the same since 1981, continuous modifications and improvemreagents and equipment is resulting in the synthesis of ever-longer, pure and inexpensivegonucleotides. There are also new methods being proposed that may, ultimately, result in aallenge to the undisputed superiority of the currently supreme phosphoramidite method. The usoligodeoxynucleotides are expanding all the time, and there is increasing interest in the synth

ribonucleotides, driven by the discovery of small noncoding RNAs and the practical applicatiRNA interference. We have come along way since the synthesis of a dimer in 1955, with syntha 100-mer routine and a 200-mer not impossible. And all of this while prices are at rock bottocertainly reassuring to know that of all the components of a PCR assay, the synthesis of angonucleotide is the least likely building block to cause a problem.

ferences

Michelson A.M., Todd, A. R. (1955) Nucleotides part XXXII. Synthesis of a dithymidinenucleotide containing a 3':5'-internucleotidic linkageJ. Chem. Soc. 2632-2638

p://pubs.rsc.org/en/content/articlelanding/1955/jr/jr9550002632


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Khorana, H. G., Buchi, H., Caruthers, M. H., Chang, S. H., Gupta, N. K., Kumar, A., Ohtsuka, aramella, V. and Weber, H. (1968) Progress in the total synthesis of the gene for alatRNA. Coring Harb Symp Quant Biol33:35-44


Agarwal, K. L., Buchi, H., Caruthers, M. H., Gupta, N., Khorana, H. G., Kleppe, K., Kumar, A

tsuka, E., Rajbhandary, U. L., Van de Sande, J. H., Sgaramella, V., Weber, H. and Yamada, T970) Total synthesis of the gene for an alanine transfer ribonucleic acid from yeast. Nature22


Khorana, H. G., Agarwal, K. L., Buchi, H., Caruthers, M. H., Gupta, N. K., Kleppe, K., Kumasuka, E., RajBhandary, U. L., Van de Sande, J. H., Sgaramella, V., Terao, T., Weber, H. and

mada, T. (1972) Studies on polynucleotides. 103. Total synthesis of the structural gene for annine transfer ribonucleic acid from yeast. J Mol Biol72:209-217


Ohtsuka, E., Ikehara, M. and Soll, D. (1982) Recent developments in the chemical synthesis olynucleotides. Nucleic Acids Res10:6553-6570Beaucage, S. L. and Caruthers, M. H. (1981) Deoxynucleoside phosphoramiditesA new cla

y intermediates for deoxypolynucleotide synthesisTetrahedron Letters22:1859-1856p://www.sciencedirect.com/science/article/pii/S0040403901904617

Caruthers, M. H., Beaucage, S. L., Becker, C., Efcavitch, J. W., Fisher, E. F., Galluppi, G.,ldman, R., deHaseth, P., Matteucci, M., McBride, L. and et, a. (1983)oxyoligonucleotide synthesis via the phosphoramidite method. Gene Amplif Anal3:1-26


McBride, L. J. and Caruthers, M. H. (1982) An investigation of several deoxynucleosideosphoramidites useful for synthesizing deoxyoligonucleotidesTetrahedron Letters24:245-2

p://www.sciencedirect.com/science/article/pii/S0040403900813763Berner, S., Muhlegger, K. and Seliger, H. (1989) Studies on the role of tetrazole in the activatphosphoramidites. Nucleic Acids Res17:853-864p://www.ncbi.nlm.nih.gov/pmc/articles/PMC331708/pdf/nar00212-0033.pdf Temsamani, J., Kubert, M. and Agrawal, S. (1995) Sequence identity of the n-1 product of a

nthetic oligonucleotide. Nucleic Acids Res23:1841-1844


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p://www.ncbi.nlm.nih.gov/entrez/query.fcgi?d=Retrieve&db=PubMed&dopt=Citation&list_uids=7596808 Hecker, K. H. and Rill, R. L. (1998) Error analysis of chemically synthesized polynucleotide

otechniques24:256-260hapter 3


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ntroduction

e previous narrative rather implies that the PCR is a fairly simple process and only requires ambination of a DNA template, two oligonucleotide primers, a dNTP mix, a simple buffer (50 mCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 8.3), a thermostable polymerase and a thermal cycler

ow the amplification of any known DNA target. As is often the case, this is true, but it is alsompletely wrong. Nature favours simplicity, and since PCR is nature in action, it is not surprisin

t its concept is not simply ingenious, but also simple. However, nature is not bent on absoluteecificity and makes use of mistakes that occur during replication to increase diversity. In additenzymatic reactions always proceed under optimal conditions and a polymerase never pauses

unt copy numbers before proceeding with its synthesis. Most obviously, cells replicate and repir nucleic acids without having to denature them.

vitro, on the other hand, this is clearly not the case and the success of a PCR reaction dependsine balance between these components, any one of which can change the outcome of a PCRction. These must be chosen according to the aims of the individual PCR experiment, individu

timised and balanced with all other components of the reaction setup. Hence it is worth reviewse in some detail, since understanding their contributions will help with initial PCR assay des

d optimisation as well as later troubleshooting.

NA template

PCR reaction must cope with a wide range of target template copy numbers that can range fromst numbers,e.g.tens of millions during an acute viral infection to samples with zero copies ofget but vast numbers of other DNAs. In general, it is advisable to add as little DNA as is feasi

a PCR assay, since too high a concentration may lead to poor results due to inhibition of andspriming by the DNA polymerase. However, this can pose an obvious problem when the aim iplify very low copy number targets since the target DNA template will comprise but a smallrcentage of the total DNA in a sample. Conversely, when a sample is made up of very dilute Dplification may be impeded by its adsorption to plasticware, by the increased risk of degradatby non-specific primer annealing resulting in false positive results. In addition, contaminationses a constant threat.

chnical issues such as DNA quality, which refers to purity (i.e. the absence of inhibitors) as w

integrity have a significant effect on the reliability of a PCR reaction[1]and must be addressedng rigorous standard operating procedures. DNA extraction procedures affect DNA quality[2]racting DNA from a tissue culture cell is obviously not comparable to extracting DNA from avironmental sample containing bacterial or fungal spore. One is rather straightforward; the oths the serious potential for a failed or inefficient DNA extraction leading to the reporting of a fagative result.

hibition

mpounds that inhibit Taqpolymerase enzyme activity are potentially present in many samples


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eme can inhibit PCR amplification of target DNA in samples containing blood[3, 4]. Faeces hav

ng been a valuable reservoir for PCR analysis[5], yet the breakdown products of haeme, such a

irubin, as well as bile salts can inhibit the PCR[6]. In addition, many of the reagents used to

tivate microorganisms, to stain cells or to prepare samples for PCR can inhibit the reaction[7,

any sources of inhibition are chemically ill defined; e.g. humic substances are a mixture of comlyphenolics produced during the decomposition of organic matter, are ubiquitous in soil and w

d may be co-purified with any material obtained from environmental samples [9]. DNA extracti

m plants has to cope with these as well as with polysaccharides, which form complexes with come bound to the DNA and inhibitTaqpolymerase[10]. Finally, it has been known for a veryme that components of the reverse transcription reaction are important sources for impurities th

hibit the PCR reaction following cDNA synthesis[11-15].

ese are just a few in a long list of components that may act as, sometimes inadvertent, PCRhibitors or enhancers, and directly affect PCR results. Critically, it appears that different PCR

ctions have differential susceptibility to inhibitors[16]. An assessment of inhibitors copurifiedring the extraction of DNA from urine revealed that susceptibility to inhibition was highly vari

tween reactions. There was no obvious explanation why one reaction should be more susceptiinhibition than another, although a possible association with amplicon GC content was noted. s serious implications for any PCR-based gene expression studies, including those using PCRays, as well as for PCR-based molecular diagnostic assays. It is not safe to assume that differeR reactions are equally susceptible to inhibition by substances co-purified in nucleic acid extr

d it is essential to perform routine quality checks on all samples, particularly if they have beenracted from anywhere but a tissue culture environment.

egrity

e integrity of the target sequence is another important parameter that affects the accuracy of qPays and is often overlooked when targeting DNA. DNA damage is not always predictable andult in false negative results, particularly critical when quantitative data are used in a clinicalting. The importance of correctly assessing DNA integrity is emphasised by the contradictoryults obtained when assessing DNA integrity as a biomarker for monitoring minimal residualease or response to therapy in cancer. Some reports suggest that plasma DNA integrity may be

reased in cancer patients[17-19]or associated with therapy response in breast cancer[20], wher

hers find no such evidence[21, 22]

.antitative results depend on the number of intact, amplifiable target sequences in the sample, w

n be affected by DNA damage that occurredin vivoor during sample collection, transport, stod processing. In most cases there is probably insufficient DNA damage to interfere with qPCRults, but this is not so for some conditions,e.g.DNA extracted from stained microscope slidem archival material. One way to determine the proportion of target sequences that are amplifiao use a method that assumes that if the DNA lesions preventing amplification occur randomly,Poisson distribution will describe their number in a given length of DNA. The mean number o

ions per base then provides a simple measure of DNA integrity, allowing the calculation of heplifiable fraction of a target sequence from this number and the target length[23].


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traction efficiency

e use of spike-and-recovery controls can be useful for identifying concerns regarding extractioiciency although, as always, it is important to choose appropriate ones. The surrogate must besent from the native sample, co-concentrate with the target of interest, lyse with equal effectivempared to target cells, and contain DNA that is extracted and recovered with efficiency equiva

that of targeted cells[24]. Since this can be difficult to achieve, some researchers add naked spNA to their sample just prior to extraction; however, this may become degraded and lead to

ccurate and variable evaluation of DNA extraction efficiency. Conversely, if the spike is addelowing the extraction process, it can no longer serve as an extraction control, although it can bed for monitoring of inhibition. Once extracted, detection becomes an issue, since an additionamer pair will be required to detect the spike. If carried out as a separate reaction, further costurred; if carried out as a dual- or multiplex rea

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