Never break the chain

Toby Hampshire discusses how to enhance QPCR results by minimising variables and selecting reagents that offer optimal performance.

QUANTITATIVE polymerase chain reaction (QPCR), also referred to as real time PCR, is used for the amplification and simultaneous quantification of target DNA or cDNA, using fluorescence detection chemistry. DNA is quantified as it accumulates in the reaction in real time, after each amplification, by measuring the amount of fluorescence emitted from a fluorescent marker in the sample. The amount of DNA present in the sample can be determined by comparing the normalised fluorescence of known standards against the PCR cycle number.

Readings are taken during the exponential phase of the reaction (the linear phase when plotted on a logarithmic scale), when there is no bias from limiting reagent concentrations. The cycle number at which the threshold for detection of fluorescence (above background) is reached is referred to as the Ct.

One-Step Reverse Transcriptase PCR can also be performed in real time (QRT-PCR). This technique offers the most sensitive and convenient method of quantifying gene expression.

Sensitivity of detection and reaction consistency are extremely important in the quantitative measurement of PCR products. This can be optimised in the selection of appropriate QPCR consumables, from plastics to reagent components, in order to reduce many of the variables inherent in the QPCR process.

One way in which the sensitivity and consistency of QPCR can be enhanced is to optimise the reflection of the fluorescent signal. This can be achieved through the use of opaque white polypropylene plates, such as Thermo Scientific ABgene opaque white PCR plates, instead of the traditional natural (or clear) plates.

When using natural or clear plates, the fluorescent signal passes through the well wall and hits the Peltier block beneath. Inconsistencies on the surface of the block reflect the signal inefficiently and, as the reflected fluorescent signal passes back through the clear plastic, it is refracted again. As a result, variations in the data between replicate wells may be due to this inefficient reflection/refraction of the signal rather than any true differences between the samples.

By contrast, white polypropylene plastic plates reflect the signal back to the detector more efficiently than any other colour (Figure 1). This increased reflection means that a true, above-baseline fluorescent signal is observed at an earlier time-point, resulting in lower Ct values and higher reaction sensitivity (Figure 2). Opaque white plastic also eliminates the problem of cross-talk between wells, further enhancing the accuracy of results.

For high throughput laboratories using automation and robotics, results can be further improved by using ultra rigid, opaque white PCR plates, such as Thermo Scientific ABgene SuperPlates. 

The high temperatures used during thermal cycling can sometimes cause conventional PCR plates to warp, making them difficult to fit into plate carriers. Other processes, such as centrifugation, may cause plates to become wedged in plate carriers. Such problems can result in sample loss, during attempts to free plates, and interruptions in workflow.

SuperPlates combine frame design with a specially selected polymer to virtually eliminate the plate warping and handling problems seen with traditional PCR plates, whilst still delivering maximum QPCR sensitivity.

Figure 1. White plates are shown to reflect the signal from fluorescein more effectively, resulting in greater sensitivity

Whilst the use of white plastic helps to return the fluorescent signal back to the QPCR detectors more efficiently, it is harder to see colourless reagents within the individual wells. This makes it more difficult to see which wells have been filled and to assess the accuracy of pipetting.  Interruptions during manual pipetting can easily lead to the omission or double dispensing of an entire row, causing highly discrepant results.

This problem can be overcome by using a coloured master mix. ABsolute Blue is the first range of master mixes to contain an inert blue dye, which allows the reagent to be easily visualised within white plastic plates and makes verification of accurate dispensing quick and easy. This visualisation of the master mix reduces the likelihood of pipetting errors and, therefore, increases the accuracy and reproducibility of results. A simple glance is all that is required to ensure that the correct amount of master mix has been added and that no wells have been omitted.

It has been demonstrated that the inert blue dye in ABsolute Blue does not have any detrimental effect on the performance of the QPCR reaction2. As a result, it has been adopted routinely in many laboratories in preference to a clear QPCR mix.

The quality and type of reagents used in QPCR master mixes can also affect the sensitivity, specificity and reproducibility of reactions. A QPCR master mix should contain a hot-start DNA polymerase to eliminate non-specific priming, which may occur before the initial denaturation step using a standard DNA polymerase. A hot-start DNA polymerase is designed to remain ‘switched off’ until the activation temperature is reached.

Figure 2.  GAPDH amplification using 2ng human genomic DNA SYBR® Green Mix in natural plates (blue) and in white plates (red). Detection above baseline occurs approximately 1-2 Cycle Thresholds sooner in samples amplified in white plates

Some suppliers use an antibody-modified Taq polymerase in their QPCR mixes. This type of modification is relatively unstable (antibodies denature at about 60°C, which is close to the annealing temperature of some primers) and there is a risk of antibody disassociation at sub-optimal temperatures, causing the Taq polymerase to begin polymerisation. At these sub-optimal temperatures, the specificity of primers can be very low and promiscuous binding (mis-priming) can occur. This non-specific priming can generate erroneous products that are further amplified in subsequent PCR cycles. The generation of these non-specific products consumes components required for PCR, drastically affecting the amplification efficiency of the target sequence.

This problem can be overcome by using a master mix that contains a chemically modified hot-start enzyme, such as the ABsolute QPCR and ABsolute Blue QPCR master mixes. Chemically modified enzymes are activated at higher temperatures than antibody-inhibited enzymes, eliminating non-specific priming and ensuring that only target DNA is amplified. 

The proprietary, chemically modified DNA polymerase used in ABsolute QPCR mixes requires an activation step at 95°C for 15 minutes (although a range of temperatures and times can be used, depending on requirements1). The enzyme remains completely inactive until it is ‘switched on’ by this high temperature incubation. To protect the enzyme at the high temperatures required for activation, a proprietary buffer is supplied with the enzyme. This ensures a maximal response from the enzyme once it has been activated, resulting in higher yields than those obtained with systems based on chemical modifications alone.

Figure 3.  Amplification of alpha-tubulin from human genomic DNA.  A 10-fold dilution series from 250ng-0.25ng DNA.  The efficiency of the dTTP reaction (blue) is 99.7% and of the dUTP reaction (red) is 94.7%

For extra specificity, an incremental activation can be used, where the enzyme is activated in steps according to the user’s wishes. This type of stepwise activation, such as 2 minute incubations at 95°C for the first 7–8 cycles, ensures a highly specific reaction where the amount of active enzyme is increased during the reaction. This is not possible using an antibody-based hot start enzyme.

Increased reaction efficiency can also be achieved by using deoxythymidine triphosphate (dTTP) in the dNTP mix instead of deoxyuridine triphosphate (dUTP). 

Traditionally, dUTP has been used in conjunction with the enzyme Uracil-N- Glycosylase (UNG), which is added to remove any amplicon carry-over contamination from previous reactions. However, with good laboratory practice, the risk of contamination can be virtually eliminated, making UNG digestion redundant.

It has been shown that the use of dUTP can cause QPCR reaction efficiencies to decrease by 5% (Figure 3). The dUTP forces the Taq polymerase to move more slowly along the template, which can increase incorporation mistakes whilst copying the amplicon and decreases the overall efficiency of the reaction. Since dTTP is a naturally occurring base, it is more readily incorporated into the amplicon, thereby increasing the reaction efficiency.

The choice of consumables used in QPCR can dramatically affect the sensitivity and consistency of reactions. By selecting products to reduce variables, increase sensitivity and improve reaction efficiency, it is possible to achieve consistent end-point readings and lower Ct values.