PCR Optimization and Troubleshooting Guide

Optimizing Strategies for PCR Amplification Success

PCR is a powerful technique used to amplify a particular region of DNA so that it can be easily detected and manipulated. Because of the great sensitivity of PCR, it has gained widespread popularity with a wide range of applications including gene cloning, DNA sequencing, mutagenesis, genotyping, assessing the expression level of a gene, and detection and diagnosis of pathogens. Despite being a powerful tool, it is not an exact science.

Quite often, despite using an established protocol to amplify a specific region of DNA, the use of the same protocol for a different DNA region can result in failed amplification, even under optimal conditions. Alternatively, amplification is successful but can lead to the generation of multiple, non-specific products, sometimes even excluding the desired product. At this point, several parameters can be altered to enhance primer-template fidelity and product yield.

While it is a good practice to optimize all the parameters in developing a new PCR protocol for the amplification of a new target, it might not be necessary or feasible to test all these variables each time. However, there are merits in undertaking some minimal optimization steps that can improve yield and reproducibility, reduce non-specific amplification, as well as give a brighter product band with minimal background following gel electrophoresis. The best condition is determined empirically but there are situations where the variables are interdependent (such as varying the amount of dNTPs can affect the concentration of Mg²⁺ in the reaction). In this guide, we highlight various optimization strategies as well as help you identify the source of any problems affecting the success of your PCR amplification.

Primer

A lot goes in designing a primer but here we summarize a few things to keep in mind:

• Aim for a Tm (melting temperature) of 65-70°C for the region of hybridization and the Tm of the primers used being no more than 5°C different from each other. Tm can be calculated using this formula: Tm = 4(G + C) + 2(A + T)°C; there are many Tm calculator and primer design tool a quick internet search away.
• Primer composition and priming sites can have dramatic effect on PCR performance. Design your primers to be 15-30 bp long with the longer lengths allowing for higher specificity. Restriction enzyme sites can be added to the 5’ primer ends making it convenient for cloning. If possible, make the primer ends with C or A as this will help with specificity.
• The GC content of the primer should be between 40-60% for best PCR amplification. Try to avoid long stretches of the same nucleotide as clusters can cause non-specific binding.
• Use an annealing temperature of about 10-15°C lower than the Tm. For best results, do a gradient PCR using the primer with the template of interest in which you vary the annealing temperature by a range of degrees above and below the Tm. It is highly important to have one product if the primer is to be used for qPCR reactions.
• Make sure the primer doesn’t bind to other regions of the template or is complementary to itself or the other primers in the reaction mix, as this will result in multiple products or primer dimerization/hairpin formation.
• A lower primer concentration in the reaction will generally result in a cleaner product. Elevated primer concentration doesn’t necessarily increase the product yield and can result in creation of primer-dimers, non-specific binding, and generation of nonspecific products. Conversely, the amplification of shorter target sequence can benefit from having a higher primer concentration if a desired amount of amplified PCR product is required. Typically, the primer concentration used is between 0.1 and 1 µM of each primer.

DNA Template

The concentration of template to use in a PCR reaction should be considered in context of the quality of the DNA as well as the number of cycles in the reaction. Using too much DNA template can lead to off-target primer annealing as well as poor DNA synthesis. Varying the number of cycles in the reaction can improve your PCR product yield. [Note: The quantified DNA concentration may be higher or lower depending on contamination, such as dust or other impurities, or degradation. To avoid this, use autoclaved tubes, wear gloves, use PCR-grade water, and work in a clean environment.]

Taq DNA Polymerase

Different versions of Taq DNA polymerase may have different specific activity and buffer requirement. A titration can be performed by varying the amount of DNA polymerase in order to find the optimal amount to be used. It should also be noted that some thermostable polymerases have proofreading activity which lowers its processivity and effect yields. In this case, higher amounts will be needed for successful amplification. Furthermore, a number of strategies (Hot-Start PCR, Time Release PCR, touchdown PCR, etc.) have been developed using temperature-based control of primer annealing to improve the specificity of primer annealing and successful amplification of the desired product; however, addressing these is beyond the scope of this guide.

dNTP

The concentration of dNTP used depends on the length of DNA to be amplified. Longer DNA fragments will require higher dNTP concentrations. However, too high a concentration can inhibit the PCR reaction. Suboptimal dNTP amount also leads to incomplete elongation. The typical dNTP concentration used is between 40 and 200 uM for each of the four dNTPs.

MgCI²

Mg²⁺ serves several functions in the PCR reaction: it is an essential cofactor for DNA polymerases, stabilizes dsDNA, and elevates the Tm. Too much Mg²⁺ leads to reduced Taq polymerase fidelity and increased nonspecific products. Too little requires stricter base pairing between the primer and DNA template and results in lower product yields. Normal MgCl₂ concentrations used are between 0.5 and 5 mM; however, the concentration may need to be adjusted if the dNTP concentrations are altered as dNTPs sequester Mg²⁺ ions.

Cycle number and length

In general, the cycle number should be kept to the minimal number needed to generate sufficient product required for further analysis. It might be better to take measures to optimize the reaction than risking nonspecific product accumulation. Furthermore, PCR specificity can also be improved by decreasing the time it takes to move between temperatures during cycling. Newer thermocyclers have significantly improved the ramping rates, are better at maintaining the correct temperature, and, consequently, reduce the time to PCR completion. This last point is important for hardworking laboratory personnel that want to amplify their gene of interest and set up further analysis or manipulation steps on the same day.

Troubleshooting Common PCR Issues for Effective PCR Reactions

Generally, if the PCR reaction didn’t work, then it could be that something was accidentally omitted from the reaction. It may be worth trying the reaction again under the same condition. It is also a good practice to run positive and negative controls with each PCR amplification to ensure that all reagents were added and are still functioning. The table below provides further guidelines for troubleshooting some of the more common PCR problems.

Problem	Possible Cause	Things to Try
No PCR product	Problem with reagents	Re-run the reaction as a reagent may be left out. Use a checklist to ensure that all the reagents were added. Check the concentrations of all reagents and, when possible, make master mixes to minimize pipetting error. Use a positive control to ensure the reagents are functional. Check that the reagents are thawed and mixed thoroughly before use. Use a new vial of dNTP solution as repeated freeze-thaw cycle may have damaged your dNTP solution. Use a different polymerase.
	Suboptimal reaction conditions	Optimize the Mg2+ and dNTP concentrations. Optimize the annealing temperature by running an annealing temperature gradient of 5-10⁰C above and below the Tm.
	Insufficient number of cycles	Increase the number of cycles. Sample the reaction by removing a small aliquot every 5 cycle to determine the correct number of cycles.
	Incorrect annealing temperature	Calculate the primer Tm and run an annealing temperature gradient with the annealing temperature range of 5-10⁰C above and below the Tm.
	Poor primer design	Reconsider your primer design and follow our primer design recommendations in the previous post. Consider increasing the primer length. Make sure the primers are non-complimentary, both internally and with other primers in the reaction.
	Template quality or quantity	Analyze DNA template quality by agarose gel electrophoresis. Too little template or template contaminated with PCR inhibitors will result in lack of product. Use a spectrophotometer to check the quantity and quality (260/280 ratio) of template DNA.
	Primers are not working	Check that the primers are diluted and used at the correct concentration. Verify the primer sequence is correct. Increase primer concentrations or redesign primers if necessary.
	Too much or too little starting template	Remake DNA template. Old templates stocks may have degraded. Dilute the template DNA and set up a reaction with a varying amount of template DNA (10-200 ng DNA range). This will determine if more template is necessary and dilute out any inhibitory substances.
	PCR inhibitors are present in the template DNA	Verify that you can amplify another region from the template DNA. If yes, then the problem lies elsewhere. Set up a reaction with varying the amount of template DNA so as to dilute out any inhibitory substances. Autoclave tubes and tips to eliminate biological inhibitors. Use fresh reagents and new tubes.
	Insufficient denaturation or difficult templates	Incubation at 95⁰C for 5 min should be sufficient to denature the template. For GC-rich sequences, use a polymerase buffer intended for GC-rich templates or add components that enhance denaturation and destabilize the DNA duplex.
	Extension time too short	The general rule is to use 1 min extension time for each kbp of target to be amplified. Increase extension time by 1 min if no product is observed.
	Thermal cycler malfunction	Verify that your thermal cycler is reaching the preset temperatures. Also, your positive control PCR reaction will determine if there was any problems with the setting up the thermal cycler such as using the wrong temperatures or times.
Incorrect Product Size (general)	Cross-contamination	Use positive and negative controls. Use barrier pipettor tips. Work in a dedicated work area. Wear gloves during reaction setup.
Long nonspecific products	Primers annealing to an off-target site on the template	Decrease the annealing and/or extension time. Decrease the extension temperature to 62–68⁰C. Increase the annealing temperature. Use less primers, DNA template, and/or polymerase. Check that the primer doesn't binds to off-target sites by BLASTing your primer sequence. Vary the Mg2+ concentration while keeping the dNTP concentrations constant.
Short nonspecific products or‘primer dimers’	Primer dimers	Increase the annealing temperature and/or time. Try to find the optimal annealing temperature using a gradient PCR machine.
	Mispriming	Increase the extension time and/or temperature to 74–78⁰C. Use less primer or Taq polymerase. Increase template DNA concentrations. Use hot-start PCR protocol. BLAST primer sequence to verify that primers have no additional complementary regions within the template DNA.
Multiple Products generated	Primer annealing temperature too low	Increase annealing temperature. Try to find the optimal annealing temperature using a gradient PCR machine.
	Too many cycles	Reduce the number of cycles used.
	Poor primer design	Verify that primers are non-complementary, both internally and to each other. Increase the length of the primer. Avoid GC-rich 3' ends.
	Excess primer	Optimize the primer concentrations.
	Incorrect template concentration	Use a spectrophotometer to check the quantity and quality (260/280 ratio) of template DNA.
	Premature replication	Set up reactions on ice using chilled components and add samples to thermocycler preheated to the denaturation temperature. Use hot-start protocol.
Faint Product Band	Extension time too short	The general rule is to use 1 min extension time for each kbp of target to be amplified. Increase extension time by 1 min if no product is observed.
	Insufficient number of cycles	Increase the number of cycles. Sample the reaction by removing a small aliquot every 5 cycle to determine the correct number of cycles.
	Template quality or quantity	Analyze DNA template quality by agarose gel electrophoresis. Too little template or template contaminated with PCR inhibitors will result in lack of product. Use a spectrophotometer to check the quantity and quality (260/280 ratio) of template DNA.
	Additive required	Add a PCR enhancer compound
Smeared Product Generated	Extension time too long	Reduce the extension time in 30 s to 1 min steps
	Denaturation temperature too low	Check that the denaturation temperature is reached and, if necessary, increase the denaturation temperature in 1⁰C steps.
	Too much template	Reduce the template amount (testing a number of dilutions).
	Too much enzyme	Reduce the amount used.

Troubleshooting Common qPCR Problems for Reproducible qPCR Results

Real-time polymerase chain reaction (qPCR) is an extremely sensitive assay in which the slightest variation can influence the results. Thus, to have a consistent and efficient qPCR assays, several parameters must first be optimized or taken into account. Below are three common problems and solutions to getting reproducible qPCR results:

1. Problem: Inconsistent results

a. Consistent Cell Growth: Due to the reliability and sensitivity of qPCR assays, this assay has been adopted as the method of choice for a wide range of applications including molecular diagnostics, genotyping, and gene expression studies. For the latter assay, the health of the cells or sample can have a tremendous effect on the variability or reproducibility of the results. As such, the pattern and timing of gene expression can vary from experiments to experiment if the condition of cell growth causes cell stress or inconsistency in growth. One way to have a consistent growth is to use quality tissue culture ware such as our TPP dishes and plates. Our line of TPP products are made from the highest purity plastics and are innovatively modeled to improve generation of consistent and reproducible data. These dishes offer a flatter, more consistent growth surface, even temperature distribution, and reduced evaporation by having stacking air vents. Try out a sample and see for yourself.

b. Evaporation problems: It is important to make sure that your plates or tubes are properly sealed in order to prevent evaporation. If the event of evaporation, the signal from the reporter dyes will increase due to an increase in signal concentration which will influence the assay results. Depending on the extent of the evaporation, you may get useable results but there will be variation from assay to assay. If you are experiencing evaporation, here are some way to minimize it: Make sure the right cap or sealing film is paired with the right plate and tubes, (2) make sure the seal properly covers all wells including the edges of the plate where evaporation most often occurs, and (3) use a roller or straight edge to thoroughly seal the adhesive cover to the plate. Check out our inventory and find the right PCR and qPCR sealing film for your PCR needs.

c. Use quality chemical and consumables: Due to the sensitivity of the assay, the slightest mistake in reaction composition or pipetting can throw off the final results. It is for this reason that most use ready-to-use reaction master mix to minimize the number of pipetting steps, laminar flow clean bench, calibrated pipette, and low binding barrier-tips to prepare their samples. It is also best to use low-binding plastic-ware (PCR tubes, strips, and tips) to minimize sample loss and increase consistency across samples and experiments. Also, the use of barrier tips and clean benches reduce the chance of contamination.

2. Problem: No signal or low signal

a. Use quality DNA template: The old adage “garbage in garbage out” applies. This is why several control reactions are included as quality control check for every reaction. Be sure to use quality DNA extraction and reverse transcription kits for a clean sample preparation, free of any PCR inhibitors. Imperfect purification of nucleic acid can leave trace amount of substance that can inhibit or interfere with PCR reactions.

b. Use validated primer and probe sequence: There are several tools available online that can assist with assessing primer and probe properties. Be sure to check the literature (and RTPrimerDB database) for validated or published probe and primer sequences. Be sure to also follow best practices when it comes to choosing target sequences such as avoid using regions having secondary structures, single nucleotide repeat regions (>4), and high GC content regions; aim to amplify a short region (75-200bp), because short sequence are amplified with higher efficiency, but long enough for the product to be distinguish from any primer-dimer formation.

c. Adjust the sensitivity of detection in your instrument: Each instrument has a specific sensitivity and dynamic range. Some instrument can detect as low as one copy of the gene while others are less sensitive. The sensitivity also effects the signal-to-noise ratio (ratio of specific hybridized fluorescent signal to nonspecific hybridization event) in which the higher the ratio, the greater the sensitivity of the instrument. Consequently, if you don’t see any peaks for your qPCR assay, then perhaps the threshold is set too high in which the lower peak signals are disregarded as noise.

3. Problem: Errors in quantification

a. Data Analysis: While qPCR assays offers high sensitivity (with a broad dynamic range of quantification) and reproducibility, it is important to have the proper controls in place for an accurate analysis and interpretation of the results. Quantification variation can result from a variety of sources such as DNA quality, reaction efficiency, enzyme inhibition, and many other factors. It is for this reason that an internal control is employed to compare the expression of the gene of interest in relation to another gene expression.

b. Use a reference dye: If your instrument allows it, use a reference dye, such as ROX dye, to compensate for any well-to-well variation. Because the reference dye concentrations are constant during the PCR reaction, the dye signal should be the same for all wells. Although the use of a reference dye is not necessary, be sure you know how your instrument compensates for any well-to-well variations.

Lastly, if you are having difficulty with your qPCR assays or planning to start a project, let MIDSCI help you select the right qPCR enzyme for your specific thermal cycler, tubes and plates, sealing film, and barrier tips for successful and reproducible results.