PCR: Master the basics for reliable results

BrittanyCarson Brittany Carson

Polymerase chain reaction (PCR) is a classic molecular technique used frequently in many labs. The basic function of this experiment is to validate the presence or absence of a specific segment of DNA. However, the significance of this segment’s presence and overall purpose of this validation can vary depending on the experimental context. Although PCR is a simple technique, often there are issues that arise and lead to ambiguous results, false positives, or false negatives.

In brief, the PCR reaction amplifies your DNA region of interest (or does not if this region is not present) to generate an abundance of amplified product of an expected size that can be visualized via an agarose gel. This product is then identical in sequence to that of your selected DNA region.

You need the following five main ingredients for the PCR reaction:

  1. DNA: DNA preparation depends on your source, and it is important that you prepare it appropriately. For example, when preparing whole organism samples (C. elegans or Drosophila) it is good to add Proteinase K in your lysis step to digest and thus clear out the proteins in your sample, including potential DNAses. If your lab does not already have an established protocol for your DNA prep, one can easily consult the literature or check with another lab that might have one. Once purified, it may be good to check the DNA concentration. As it is an amplification protocol, a little DNA should do the trick, but my experience has shown that this depends on the particular primers, as some primers require greater DNA input.
  2. PCR primers: Primers are short strands of nucleotides that are complementary to sequences around your DNA region of interest and serve as starting points for the PCR reaction. You will need both a forward and a reverse primer. You will often have to design your own primers, and good primer design is essential for a successful experiment. The primer sequence is chosen to provide optimal primer characteristics. PCR primers should be approximately 21-25 nucleotides in length, have a GC percentage around 50%, and a melting temperature to allow for dissociation during cycles. There is software and/or many websites that can assist you in primer design so as to avoid formation of secondary structures within the primer, such as hairpins, or primer-dimers where the two dimers associate with each other and not the DNA sequence of interest. The key in primer prep and usage is to mix well (vortex) and use the appropriate concentration.
  3. Deoxynucleotide triphosphates (dNTPs): Specifically, dATP, dCTP, dGTP, and dTTP are the four bases of DNA and thus serve as the building blocks for the newly synthesized DNA segment. They are added in equal molarity, either separately, mixed, or together in a mix with the Taq polymerase.
  4. Taq: The enzyme to synthesize your PCR product. MgCl2 is also added to the mix, as magnesium serves as a cofactor for Taq activity. Your Taq choice should depend on your experimental purpose. If trying to simply verify the presence of a DNA sequence, basic Taq is sufficient. If, alternatively, you wish to sequence your PCR product, a “high-fidelity” Taq is preferable, as it leads to minimal error in sequence amplification. As Taq is an enzyme, handle on ice and try to aliquot to avoid freeze-thaw cycles.
  5. PCR buffer: A buffer to promote the PCR reaction.

Once these ingredients are combined, sterile water can be used to make up additional volume. You then need to place your samples in a thermocycler with a program set to cycle through three steps:

  1. Denaturation, allowing for dissociation of DNA and primers.
  2. Annealing, allowing for association of DNA and primers.
  3. Sequence extension.

These steps cycle several times to generate a sufficient amplified product, followed by the program cooling to 4°C as a holding temperature.

To visualize your product, prepare an agarose gel including a DNA gel stain. Once the gel solidifies, place it in a gel box apparatus, load your samples, and apply an electric field through to dissociate the PCR product according to size. The size should be predicted based on where your primers are located in the DNA sequence relative to each other. The percentage of agarose in the gel should depend on your PCR product size, with higher percentage gels (more than 1%) better for dissociation of smaller bands. Be sure to include a DNA ladder to monitor proper DNA separation and that band size is as expected. Always include a positive control reaction in your PCR to verify that the reaction worked, and that the primers can amplify the region of interest if it is present. It is also wise to perform an additional reaction without your DNA, where the DNA is replaced with water, to verify that there is no contaminant DNA in your other PCR ingredients.

Finally, if you do not see a band—whether it is a positive or negative for your experiment—you can amplify another target sequence in your DNA to confirm that the DNA is prepared accordingly for PCR—although this is a less likely culprit. Happy PCR-ing!

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Brittany Carson

Brittany Carson

I am a post doc at the Lunenfeld-Tanenbaum Institute in Toronto. I currently work on glioma, but have studied breast cancer, multiple myeloma and renal cancer, with a focus on cell-signaling pathways, translational regulation, the cell cycle and the cytoskeleton to develop novel biomarkers and therapeutic targets. My science journey has taken me through cell culture, flies, worms and mice and through many different techniques. I love photography (including microscopy), traveling and non-science reading.