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Sanger Sequencing Troubleshooting: PCR thermal cycler & PCR consumables in the spotlight!

By Dr. Stefanie Rösel Lab Academy

Weak or noisy peaks, unspecific traces or multiple sequences are unfavorable Sanger sequencing results. One look at the traces is enough to know if troubleshooting is now the order of the day. Did you know that PCR thermal cyclers and PCR consumables can contribute to success or failure of sequencing reactions? This fact is widely underestimated and rarely covered in trouble shooting guides. In this article we bring this fact into spotlight – you will learn why, how and what to do...

Sanger Sequencing

Sanger sequencing (also known as cycle-sequencing, chain-termination sequencing or dideoxy sequencing) belongs to the most popular DNA sequencing methods for deciphering the nucleotide order of a DNA. Its appreciated advantage is its consistent accuracy - even with long DNA strands. Whether for the detection of genetic diseases and pathogens, for the generation of phylogenetic trees, for the analysis of mutations, for the confirmation/control of gene editing experiments or for the verification of cell lines - Sanger sequencing is broadly used and enjoys great popularity.

The actual Sanger sequencing reaction is intrinsically an "almost" normal PCR. The major difference is the additional usage of dideoxyribonucleotides (ddNTPs). The ddNTPs are fluorescently labelled with an individual color for each base and lack the 3´-OH group that is required for strand extension. Within the PCR reaction they are randomly incorporated and cause chain terminations by chance. Strands of each possible length are produced. Afterwards, capillary electrophoresis separates the chain-terminated products according to their length in single-nucleotide resolution. The different fluorescence signals derived from the labelled fragments mark the respective base. Finally, the "traces" of the fluorescence peaks reveal the sequence of the template DNA strand in the electropherogram.

It starts with PCR…

Your first look on the electropherogram promptly shows the quality of the sequencing – good, bad or something in between. What we aim to is clear:
  • Proper read lengths for maximum information
  • Read accuracy reflected by peak-resolution, consistent peak spacings and heights and tolerable noise
  • Reproducibility between runs, i.e. the method’s stability referring to the performance of different samples within one run or the quality of different runs
…getting the maximum outcome per run and above all, no repetition!

Troubleshooting starts when the sequences leave a lot to be desired. The first starting point is PCR – as the reaction in the thermal cycler forms the basis for successful sequencing. There are many sophisticated troubleshooting guides available online which comprehensively cover diverse aspects of PCR like template, primer, etc. In addition, PCR thermal cycler and PCR consumables can likewise contribute to success or failure of the sequencing reaction. Compare it to building a house: if the foundation is not stable, the whole house is at risk of collapse. Back to PCR: cycler and consumables can really mess you up as they may affect the subsequent steps and, in the end, your result.

Failed or weak sequences

Common causes of weak and noisy peaks, no sequence data or sequences which do not match any other annotated sequence refer to template DNA, primer or the presence of inhibitory contaminants – all well described in literature. But to what extent are PCR cyclers and PCR consumables involved?

  • No or low yield due to overshoot
    Too high temperatures caused by block inertia after the heating ramp at the beginning of denaturation may impair polymerase efficiency up to no activity. This may result in loss of PCR products or no detectable amplification and, consequently, in weak peaks or no traces at all. Sequencing reactions with small volumes are particularly at risk here: your sample reaches the desired temperature faster and can then overshoot.

    Use a PCR thermal cycler that controls the temperature profiles accurately and reliably. This keeps overshoot in an optimal and controlled range, so that your polymerase can do its job.

  • Edge-effect
    Do you frequently have low-quality sequences at positions in edge areas of your microtiter plate? A typical indicator for the so-called edge effect! A bent plate or an improper fit of the sealing can lead to evaporation. This usually affects the outer rows of your PCR plate.
    Besides increased evaporation, an inhomogeneous temperature distribution in the PCR thermal cycler block might be a cause. This is attributable to the block construction and, again, mainly impacts the wells of the edge areas.

    Do whatever is necessary to avoid evaporation (see next bullet). Moreover, your PCR thermal cycler block should reliably provide a consistent temperature profile across your microtiter plate. If you check off these two items, you can exclude the edge-effect as probable cause for weak or failed sequences in edge areas.

  • Evaporation
    Whenever liquid evaporates, the concentration of the reaction components increases. Thus, you have a certain probability that the reaction will not go as desired. The effects can be diverse, ranging from no effect (if evaporation is marginal) up to no result. Again, small volumes can be particularly affected, as evaporation is proportionally more severe here.

    Secure sealing, no evaporation! Be sure, that your microtiter plates are tightly sealed, and your tubes safely locked. Use high-quality PCR plates which are stable and do not bend under high temperatures. Use tight sealings like heat sealings. The lid of the PCR thermal cycler should fit tightly. A heated lid prevents liquid condensation on the vessel sealing.
    The smaller the volumes, the more evaporation protection is necessary!

  • Unequal heat-transfer in PCR consumables
    Some samples perform worse than others? Vessel walls with varying thicknesses (within the same plate) may cause an uneven heat-transfer into the samples. Vessels with thicker walls may take longer to heat up, vessels with thinner walls are heated up faster. For the first issue, the temperature might not reach the necessary denaturation temperature on time, which affects denaturation of the double-strand – the prerequisite for primer annealing. The latter issue may cause an overshoot.

    Ensure to use PCR consumables with equally thin vessel walls for fast and equal heat-transfer.

  • PCR-inhibitors
    Leachables like slip agents or plasticizers can be released out of your PCR plates or tubes. They inhibit the enzymatic reaction. Likewise, undesired DNases can degrade your template DNA.

    Use high-purity consumables which are free of DNases, PCR-inhibitors or leachables.

Multiple sequences

Common causes of overlapping peaks can be contamination with other templates, accidently mixing of different sequencing reactions or even wrong primers - to name just a few. They are also comprehensively covered in many troubleshooting guides. Causes related to thermal cycler and PCR consumables can be follows:

  • Mispriming due to undershoot or edge effect
    You have no worries on your primer design, primer amount and the annealing temperature? Then it’s time to think about “undershoot”. Undershoot means that too low temperatures at beginning of annealing lower the specificity of primer annealing. The result: unspecific products.
    Do sequencing results with multiple trace signals accumulate in edge areas of your microtiter plate? Wells at the plate edges might suffer from too low annealing temperatures caused by temperature differences across the plate and between the wells. Your primers decided to do their own business, anneal with a lower specificity and generate misprimed secondary products.

    Just as described above (see Chapter “failed or weak sequences”), the block is the culprit. PCR thermal cycler blocks with an excellent temperature homogeneity ensure a stable temperature profile across the plate. An adequate temperature control keeps undershoot in an optimal range - so that your primers do what they are supposed to do.

  • Mispriming due to unequal heat-transfer in PCR consumables
    It may take for some “disadvantaged” samples longer to heat up. Consequently, the annealing temperature might not rise fast enough and weakens the primer annealing efficiency.

    One more argument for using PCR consumables with equally thin vessel walls

  • Contaminated PCR consumables
    Undesirable DNA causes undesirable sequences.

    PCR-clean consumables which are free of DNA.

The enzymatic reaction in the thermal cycler forms the basis for successful sanger sequencing. Conclusion: Build a proper foundation with a reliable, high-quality thermal cycler and appropriate PCR consumables.
But that's not the end of the story! All issues mentioned here have one thing in common: they can impair reproducibility. Read what’s behind in part II of our Sanger Sequencing Troubleshooting article series…

Be one step ahead in troubleshooting!Check out Eppendorf’s PCR solutions: www.eppendorf.com/pcr