47747_LM_PCR AND qPCR_eBOOK_JL (5)
? Questions to Ask When Buying PCR Technology
? Optimizing and Troubleshooting PCR
? Designing qPCR Assays
? The Latest in High-Throughput Thermal Cyclers
? Challenges of Microscopic Samples in Solution
PCR AND qPCR
RESOURCE GUIDE
Questions to Ask When Buying PCR or qPCR Technology
PCR is a technique used to amplify DNA strands by cycling through specific temperatures to facilitate different stages of the reaction. Building on this, qPCR allows for the quantita- tion of DNA strands in real time. When purchasing technolo- gy for PCR and qPCR, consider the accuracy of the tempera- ture and fluorescence measurements, and the robustness of the instrument as it will be running for long periods of time.
What type of equipment will you need?
Maintenance tip
Verifying the temperature within your PCR/ qPCR technology once or twice a year is crucial to maintaining the accuracy and uniformity within the heat block. Inaccurate readings can impact your experimental results and lead to lost samples and high downstream sequencing costs
Traditional PCR is used in preparation for sequencing and assays and as a qualitative endpoint, showing the presence or absence of amplified product. Quantitative PCR (qPCR) measures relative DNA amplification in real time based on standards included in the reaction mix. Digital PCR (dPCR)
uses statistical algorithms to determine the absolute quantity of DNA in a sample matrix without the use of standards or references. Different instruments are required to conduct each type of PCR.
What sample formats will you be using?
Thermocyclers come with a variety of plates capable of accommodating 0.2 mL tubes, 0.5 mL tubes, 96-well plates, or 384-well plates. Look for instruments with interchangeable plates and make sure you purchase an instrument with plates that are able to accommodate your sample vessels. For qPCR,
purchase tubes and plates that are optically optimized for your instrument as this type of PCR relies on light/fluores- cence to quantify DNA. The wrong tubes or plates can affect the light detected by the instrument and skew your results. Check your user manual for manufacturer recommendations.
What are your throughput requirements? How many samples can the instrument run at once?
Systems with faster ramp up or cool down times will be able to conduct reactions faster, increasing the number of reactions you can perform in a day and overall throughput. If more than one user wants to run reactions at the same time, look for multi-block thermocyclers that can run separate reactions with different time and temperature parameters simultaneously.
How easy is the instrument to program?
Does the instrument come with software and what are its capabilities?
How much space will the instrument take up in your lab?
PCR and qPCR Resource Guide
Optimizing and Troubleshooting PCR
Polymerase chain reaction (PCR) and quantitative or real-time PCR (qPCR, RT-PCR) share many similarities but function differently and generate divergent end products
By Angelo DePalma, PhD
While PCR simply amplifies DNA, qPCR detects and quantifies product in real time as it is being amplified, hence the “RT” and “q” designations. qPCR amplifica- tion curves generated from fluorescence detection pro- vide an accurate measure of how much material the analyst began with. By contrast, conventional or “end point” PCR results are usually visualized through gel electrophoresis, and thus give at best semiquantita- tive measurements through the use of gel standards until sequencing or further analysis via other means.
At the end of traditional PCR, you will typically have a bright band on the gel regardless of how much material you started with. With qPCR, standards are used to determine what the starting amount was over a scale of many orders of magnitude.
The amplification curve is the principal indicator that a qPCR has proceeded satisfactorily—or not. It is generated by exciting the fluorescence labels and quantifying the response by application of a software algorithm to generate a curve whose shape is diagnostic.
Since it is based on fluorescence detection, qPCR is capable of detecting multiple genetic targets in the same reaction or sample and can quantify their proportions. A useful applica- tion of this capability is determining male/female or human/ nonhuman genetic elements.
General qPCR troubleshooting
In qPCR, poor amplification efficiency can be a major prob- lem. Quite a large number of factors, from divergent sources, contribute to efficiency. Protocol-based issues arise from reaction setup, design of primers and cycling steps, quantity of polymerase used, and magnesium chloride concentration. Sample quality, matrix, and isolation method also contribute.
These factors commonly result in noise or delayed amplifica- tion curves. Delays generally indicate low target concentra- tion but can be due to poor amplification efficiency. Noise is somewhat more complex, as it may arise from the amplifica- tion of unwanted targets due to poor primer design, subopti- mal temperature cycling, or other factors.
Delayed amplification curves aren’t always bad, but it’s pref- erable to achieve robust amplification where you obtain solid curves from a minimal amount of target DNA.
Troubleshooting qPCR involves testing conditions and components known to affect PCR efficiency. These include annealing temperatures, magnesium chloride and primer concentrations, and others. Operators can almost always re- solve these problems, and the experimentation is usually not resource prohibitive.
Working in a 96-well qPCR plate enables investigators to test several factors simultaneously.
Instead of using a plate reader, the troubleshooting experi- ment runs inside the qPCR instrument, which provides incu-
PCR and qPCR Resource Guide
PCR and qPCR Resource Guide
bation, temperature cycling, and fluorescence measurement. Depending on the depth of a particular issue parameter, testing takes anywhere from a few hours to several days.
Labs working on a new qPCR experiment usually begin with a master mix that includes all reagents needed to conduct gene amplification. Numerous master mixes are available commercially, suitable for any conceivable gene target and sample type. If the results are not as expected or you don’t achieve satisfactory amplification using a master mix, begin an optimization exercise.
Mind your SOPs
Depending on the primers, reagents, and target, PCRs may be more or less sensitive to reaction conditions such as salt and magnesium concentrations, or carryover ethanol from sample preparation. Labs using homebrew DNA preparation methods may see variability among tubes or wells when performing PCR. Errors can also increase as protocol rigor goes down, especially in multiplexed reactions that aim to amplify more than one target.
Experts advise analysts to institute more rigorous labora- tory practices, particularly around sample preparation, and to think consistency. Sloppy lab technique can sometimes obscure secondary sources of variability.
For labs used to making up their own reagents, trouble- shooting may be as simple as purchasing reagents that are formulated to strict concentration standards. Vendors provide consistency upfront. They test their products, so consumers don’t need to worry about reagent inconsistencies affecting their reactions.
PCR has advanced to the point where vendors now offer reagent kits containing buffers and enzymes that are less sensitive to reaction conditions, for example magnesium or
ethanol concentrations. However, users should still take care when running PCRs with these kits.
PCR is sensitive to contaminants, so operators should wear gloves, use PCR-grade water and super-clean glassware, and work inside a clean area. Environmental contaminants— acids, bases, ions—can affect PCR through inhibition of polymerase enzymes, resulting in less vigorous amplification (or “slower” curves for qPCR). Again, some reagent kits are designed to be less sensitive to contamination effects.
Advanced debugging
A common advanced problem involves sub-optimally designed primers. Primers that are not unique or that don’t bind strongly to the target gene will recognize and amplify similar sequences, decreasing reaction efficiency and adding background.
Amplification bias leads to issues in multiplex PCR. One way that bias arises is when the primer pair for sequence A is 100 percent efficient but the efficiency of the primer pair for sequence B is only 80 percent. Since PCR is a logarithmic operation, it will not take long for sequence A to swamp out the signal from sequence B. Then, it will appear as though
there is more of gene A than gene B in the sample. This effect occurs in both qPCR and traditional PCR.
Operators can overcome amplification bias by redesigning their primers for optimal target selectivity. Running each am- plification singly will shed some light on potential efficiency mismatches. If bias cannot be overcome using different prim- ers or through protocol adjustments, simply understanding the nature and extent of bias may be enough.
Primers are one of the most important components of PCR.
If you have well-designed primers, then other sources of error become less prominent.
Designing qPCR Assays
To quantify nucleic acids, DNA, or RNA, scientists often use the quantitative polymerase chain reaction (qPCR)
By Mike May, PhD
Key steps at the start simplify optimization and validation
qPCR—like all PCR methods—amplifies nucleic acids, but it also monitors the process in real time. When putting qPCR to work, scientists must design and validate assays. This poses a variety of challenges in various aspects of this process.
Key challenges when designing qPCR assays are selecting the primers and probe sequences, as well as the reporter dyes. This can be particularly difficult for multiplex assays. It can also be difficult to balance the choice of oligo sequences and detection chemistries compatible with available qPCR instrumentation. The more targets a user wants to detect in a multiplex assay, the more precarious this balance can be.
Additional challenges further complicate the design stage. For example, users should obtain the most current se- quence information and avoid sequence variation such as SNPs, insertions, deletions, and low-complexity sequences. With that information, a scientist then needs to make sure the probe will target a unique sequence and not multiple genome fragments.
Features of the target affect how well the PCR works. For ex- ample, it is best to design assays across exon-exon junctions so
that you are not detecting target genomic DNA. Users should also consider whether they want to detect a specific transcript or different transcript variants of the same gene. For trans- genic experiments, scientists should look at species-specif- ic targets.
Details of design
Some advances in qPCR technology make it easier to design new assays. qPCR instrumentation is available with multiple channels that can simplify experiment design by expanding the design possibilities for oligos and probes. In terms of re- agents, the continuous introduction of new probe chemistries can help add flexibility to assay design. For a new qPCR assay, consider using a design tool.
Optimizing the assay
Once a scientist designs an assay, there’s more work to do, such as optimizing it. For successful qPCR assays, variables such as component concentrations and thermal cycling pa- rameters must be optimized. Optimization of qPCR matrices can be very complex and should include testing of representa- tive sample material
The level of optimization needed depends on the quality of the design. Good assay design means less work when it comes to optimizing the assay. For example, primers and probes should be designed at uniform temperatures. If the design
is not constructed carefully enough, more work is required to make the assay run as efficiently as possible. If you don’t optimize your assay design up front, then work will need to
go into finding the best annealing and extension temperatures for not only your primers and probes, but also for your master mix and instrumentation.
PCR and qPCR Resource Guide
PCR and qPCR Resource Guide
The optimization can be more difficult in some situations than in others. If sample material is sparse, then availability for assay optimization experiments becomes problematic.
For the best outcomes in optimization, scientists might seek specific features in qPCR technology. For example, instru- mentation with high-throughput potential can facilitate optimization. These instruments may have fast thermal cy- cling, automation such as robotic pipetting stations, or other features to speed up the process. Running multiple thermal cycling profiles at the same time also accelerates how fast a scientist can optimize an assay.
The reagents also impact optimization. New master mix products are regularly introduced and can allow for the removal of certain parameters from the optimization matrix, such as MgCl2 or enzyme concentration. This can reduce the number of optimizations that must be performed before an assay is up and running.
Validation is needed
Lab Manager 6
After going through the steps to design and optimize a qPCR assay, it needs to be validated—to show that it really does
what it is supposed to do. One of the most challenging aspects of validation can be assessing the variance introduced by dif- ferent qPCR platforms. It can be frustrating to discover that the instrument used to develop your qPCR assay is prone to large inter-instrument variations.
To reduce the odds of such problems, select instruments with low inter-instrument variation early in the assay development. To choose such an instrument, run the assay on as least three different instruments of a specific model. Manufacturers are often helpful in putting you in touch with labs that have such instruments.
The required effort in validation often depends on the appli- cation at hand. In many cases, commercially available assays will do the job. When needed, however, completely novel qPCR assays can be developed for specific applications. By combining the right qPCR and reagents, plus experience and design tools where needed, scientists can design, optimize, and validate assays.
Optimizing PCR Experiments Infographic
Optimizing
PCR EXPERIMENTS
Primer design, reaction mixtures, primer melting temperatures, and thermal cycling conditions contribute to the success of PCR experiments. This infographic will guide you through the steps to achieve maximum efficiency when performing PCR experiments.
The Polymerase Chain Reaction (PCR) method is used to amplify target DNA sequences. Primer design, reaction mixtures, primer melting temperatures, and thermal cycling conditions contribute to the success of the experiment. Optimizing these conditions and taking steps to prevent contamination help to ensure successful outcomes.
Download the full infographic courtesy of Lab Manager
The Latest in High- Throughput
Thermal Cyclers
Both real-time and endpoint-based PCR studies can benefit from the drive toward automation with high- throughput thermal cyclers
Brandoch Cook, PhD
While the impetus for the contemporary push toward ever greater throughput and automation in the life sciences initiat- ed in coordinated efforts to sequence the human genome, par- allel strategies to identify and characterize genetic expression patterns, both in real-time and at experimental endpoints, spurred an analogous growth of throughput-driven technolo- gies that continues to proceed apace.
In the late 1990s, the first system was introduced to potentiate thermal cycling, detection, and analysis of threshold cycle values for gene expression in quantitative reverse-transcrip- tase-PCR (qPCR) assays. The ability of these systems to deliver meaningful transcript expression data is founded on the Therma-Base technology utilized in their plate-based module, which delivers a high degree of thermal stability and homogeneity across multi-well plates. The engine driving rapid and predictable temperature changes lies within a circuit board employing Peltier elements, the venerable stan- dards for most plate- and platform-based thermal cyclers.
The Peltier effect occurs when heat is absorbed or emitted at the junction between two dissimilar semiconductors through which a current is passed. A Peltier element is usually incor- porated into a conductive metal block, with a ventrally adja- cent finned sink through which heat can dissipate. Heating or cooling can be actuated and controlled via the amount and directionality of the current.
Recent developments in thermal cyclers
The initial instruments have been updated with an increased capacity from a maximum of 384-well to 1,536-well plates, a per-assay throughput improvement of four-fold. Commensu- rate with this increase, per-reaction volumes have decreased substantially, with a maximum of two microliters, versus 10 microliters for the 384-well format.
There is throughput, and then there is throughput. Although Peltier elements can deliver predictable and rapid tempera- ture ramps, the constant strain inherent in activating and reversing their potential can cause them to break down over time, and the emitted heat that must be vented can expand the functional footprint of qPCR cyclers well beyond their measured cubic allotment. Alternately, plates can be moved through multi-chambered, water-based thermal cyclers
that maintain stable temperatures in discrete baths and thus eliminate by up to 40 percent the time associated with Peltier element temperature ramps.
Several high-throughput water bath cyclers are available and can deploy 96-well, 384-well, or 1,536-well plates in rapid succession. Additionally, they can accommodate the ultimate solution for automated PCR, Array Tape, which obviates the need for plates and can be run almost continuously like news- sheet under a printing press because of its low volume and thermal mass. These are endpoint machines optimized for
PCR and qPCR Resource Guide
PCR and qPCR Resource Guide
standard PCR. As such, they are primed to take on workflows for large-scale analyses such as mapping genetic interactions or KASP assays to identify rare single nucleotide polymor- phisms in novel biomarkers.
At least one version on the market uses a circulation jet to maintain homogeneous water temperatures and eliminate the radiative edge effect that can often impact the consisten-
cy of high-throughput PCR assays. Instruments can process upwards of 145,000-230,000 samples per workday and can be incorporated into automated workflows using liquid handlers and robotic arms to transit plates or Array Tape from bath
to bath. For laboratories looking to automate their genetic analyses, there are attractive options for both real-time and endpoint assays, in configurations suitable for the benchtop.
PCR and qPCR Resource Guide
The Ins and Outs of Digital PCR (dPCR)
Absolute quantification of target nucleic acids using partitioning
Digital polymerase chain reaction (dPCR) enables absolute quantification of target nucleic acids in a sample. The sample is partitioned into many individual reactions. Following PCR amplification, amplified target sequences are detected by fluorescence and the ratio of positive to total partitions is used to determine concentration of target sequence.
ABSOLUTE QUANTIFICATION OF TARGET NUCLEIC ACIDS USING PARTITIONING
Digital polymerase chain reaction (dPCR) enables absolute quantification of target nucleic acids in a sample. The sample is partitioned into many individual reactions, each containing only a few target sequences. Following PCR amplification, amplified target sequences are detected by fluorescence and the ratio of positive partitions to total partitions is used to determine the concentration of the target sequence
in the sample. Quantification is based on the random distribution of molecules in multiple partitions that follows a Poisson distribution.
In this infographic you’ll learn more about:
sample partitioning concentrates target sequences and reduces template competition
dPCR does not rely on calibration curves for sample quantification
offers a higher tolerance to inhibitors present in samples
improved detection of low-copy number variants
SAMPLE PREPARATION
Download the full infographic courtesy of Lab Manager
PCR solution containing template, fluorescence-quencher probes, primers, PCR master mix (DNA polymerase, dNTPs, MgCl2, and reaction buffers).
PARTITIONING
The sample is partitioned into individual sub-reactions, using microfluidic chamber-based, micro-well chip-based, or droplet-based methods.
AMPLIFICATION DETECTION
Challenges of Microscopic Samples in Solution
How to maintain sample integrity when the scale of the experiment is significantly reduced
Damon Anderson, PhD
The last 20 years have witnessed the explosion of technol- ogy miniaturization. Advancements in materials science, engineering, and microfabrication have led the transfor- mation from macroscale, to microscale, and ultimately to nanoscale technologies. Micro and nanoscale technology innovations have fueled new applications across the spec- trum of biomedical and molecular medicine. Scaling down from the macro to the microscale level has major advantages for analyzing events, such as discrete biomolecular processes and interactions. However, reducing the size of materials and the volumes of solutions involved in these investigations brings challenges.
The structures and chemistry of the contacting materials influence the behavior of molecules in solution. Flow rate, pressure, temperature, and evaporation impact the integrity of solutions, which can challenge the analysis, and ultimately the quality, of the data. These considerations shed light on an interesting and important point regarding the challenges of biomolecules in solutions—how does one ensure sample integrity is maintained when the scale of the experiment is
significantly reduced? To address this question, we can gain insight from a state-of-the-art microscale technique and its important biomedical research applications.
Definitions:
Macroscale: Anything greater than ~100
micrometers in size, and visible to the naked eye
Microscale: Ranges from 100 micrometers
to 100 nanometers
Nanoscale: Ranges from 100 nanometers
to 1 nanometer, or the scale of atomic resolution
Microvolume: In this context, ranges from
100 microliters to 100 nanoliters
Nanovolume: Ranges from 100 nanoliters to 1 nanoliter
Microfluidics and digital polymerase chain reaction (PCR)
PCR technologies have successfully addressed many chal- lenges in research and medicine. Several new screening diagnostics use PCR-based liquid biopsy as a platform for de- tecting genetic patterns indicative of cancer. PCR-based diag- nostics can also be applied to genetic disorders that entail rare
PCR and qPCR Resource Guide
PCR and qPCR Resource Guide
DNA variants or single nucleotide polymorphisms (SNPs). The sensitivity and accuracy of PCR are key attributes for genetics-based diagnostics. Traditional PCR methods can struggle with complex sample backgrounds, amplification bias, and the demands of quantitation.
Digital PCR (dPCR) arose from the observations that PCR could be accomplished using a single copy of DNA. dPCR measures discrete PCR amplification reactions using single molecules of DNA, whereas conventional PCR measures the sum of many amplifications at a time in one reaction. Quan- titation of dPCR is absolute, while conventional PCR relies on relative comparison with internal standards and use of a standard curve. PCR efficiency bias, arising from preferential amplification of high-copy DNA in a sample, is eliminated in dPCR. The impact of background interferences is minimized due to the sample dilution process during dPCR analysis.
A requirement for dPCR is separation of a sample into dis- crete reactions of single DNA molecules, and the incorpora- tion of microfluidics and microscale partitioning technologies is critical. With partitioning into thousands of nanovolume droplets, digital measurements are taken and individual DNA molecules identified through yes or no results. Rare DNA sequence detection, single-cell analysis, and next-generation sequencing are all enabled by microfluidics and nanovolume partitioning in dPCR.
Instruments must partition and manipulate DNA samples for successful analyses. Microfluidic channels and partitioning within the dPCR instrument into tens of thousands of indi- vidual reactions ensures that the sample background is suffi-
ciently dilute, and free from interfering materials and other activities that challenge sample integrity. During detection, microfluidic channels allow individual partitioned samples to be analyzed and digital results to be collected and interpret- ed. This massive partitioning and detection are coupled with Poisson distribution statistics calculations to measure the absolute quantity of sample DNA.
dPCR platforms use a range of technologies to partition microvolume samples during analysis. One example involves creating a mixed sample oil emulsion during microfluidic separations. Droplets of this emulsion are deposited into nanovolume plate wells. The oil encapsulates the aqueous sample, preventing loss due to non-specific absorption or evaporation as the droplet makes its way through PCR and measurement analysis. Other technologies use micro-fabri- cated compartments placed within the microfluidic device trapping nanovolume samples. The compartment walls are made of non-binding materials to ensure minimal DNA loss through absorption. Other technologies involve DNA-specific magnetic beads, in which a single molecule of DNA bound
to a bead is encapsulated in an oil emulsion and subjected to PCR reaction.
Regardless of the method of partitioning, the goal of these dPCR technologies is the same—to stabilize and maintain the integrity of microscale samples during analysis.
Product Spotlight
PIPETMAX®: An Automated Pipetting System with Protocol Support & Training
PIPETMAX is a benchtop-sized automated pipetting solution for the efficient processing of high-throughput biological assays. This easy-to-use system helps improve accuracy,
reproducibility, and consistency among samples processed in your qPCR and PCR workflows.
The software of PIPETMAX is customizable to fit the specific needs of the assay, allowing you to configure and customize runs to any reagent, kit, or protocol through strong post-sale support
from Gilson’s team of experts. With intelligent tip management, the pipette head allows up to 8 tips
to be used simultaneously. With a volume range from 1 µL to 1200 µL, PIPETMAX is an ideal solution for not only qPCR workflows, but NGS reactions and cell-based assays as well.
LEARN MORE
Finally, the small footprint lets it easily fit on a lab bench or under a fume hood. At a weight of just under 40 pounds, it’s easy to move to the location most suited for your assays.
Gilson is a family-owned global manufacturer of liquid handling, purification, and extraction
solutions for the life sciences industry. We help researchers advance the pace of discovery by creating easy-to-use lab instruments that improve reproducibility and traceability. For decades, we’ve been developing innovative products such as PIPETMAN®. By partnering closely with the scientific community, we’re continuously advancing our product offerings and have added automated pipetting systems and software to our portfolio. Backed by worldwide R&D, service, and support, Gilson enables verifiable science to make lab life easier for our customers.
www.gilson.com
Lab Manager 13
PCR and qPCR Resource Guide
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