Manual Satis (Time Cycle Book 1)

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The development of the polymerase chain reaction PCR is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology.

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The theoretical process was outlined by Keppe and coworkers in ; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus , consequently the name Taq DNA polymerase. PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA i.

While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels.

Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem s.

This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. Designing appropriate primers is essential to the successful outcome of a PCR experiment. The 3' end of primers should contain a G or C in order to clamp the primer and prevent "breathing" of ends, increasing priming efficiency. DNA "breathing" occurs when ends do not stay annealed but fray or split apart. The three hydrogen bonds in GC pairs help prevent breathing but also increase the melting temperature of the primers.

The 3' ends of a primer set, which includes a plus strand primer and a minus strand primer, should not be complementary to each other, nor can the 3' end of a single primer be complementary to other sequences in the primer. These two scenarios result in formation of primer dimers and hairpin loop structures, respectively. Di-nucleotide repeats e. If unavoidable due to nature of the DNA template, then only include repeats or single base runs with a maximum of 4 bases. There are many computer programs designed to aid in designing primer pairs. In order to avoid amplification of related pseudogenes or homologs it could be useful to run a blast on NCBI to check for the target specificity of the primers.

When setting up a PCR experiment, it is important to be prepared. Wear gloves to avoid contaminating the reaction mixture or reagents.

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Include a negative control, and if possible a positive control. Arrange all reagents needed for the PCR experiment in a freshly filled ice bucket, and let them thaw completely before setting up a reaction Figure 2. Keep the reagents on ice throughout the experiment.

Additives are discussed further in the trouble shooting section. When setting up several PCR experiments that all use the same reagents, they can be scaled appropriately and combined together in a master mixture Master Mix. This step can be done in a sterile 1. To analyze the amplicons resulting from a PCR experiment, reagents and equipment for agarose gel electrophoresis is required. To approximate the size of a PCR product, an appropriate, commercially available molecular weight size standard is needed. Start by making a table of reagents that will be added to the reaction mixture see Table 1.

Reaction volumes will vary depending on the concentrations of the stock reagents. Add only if it is not present in the 10X buffer or as needed for PCR optimization. For example, to obtain the 4. Add 10 4 to 10 7 molecules or about 1 to ng DNA template. Add 0. Add Q. However, it should be noted that water is added first but requires initially making a table of reagents and determining the volumes of all other reagents added to the reaction.

Place a 96 well plate into the ice bucket as a holder for the 0.

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Allowing PCR reagents to be added into cold 0. Pipette the following PCR reagents in the following order into a 0. Since experiments should have at least a negative control, and possibly a positive control, it is beneficial to set up a Master Mix in a 1. In a separate 0. In addition, another reaction if reagents are available should contain a positive control using template DNA and or primers previously known to amplify under the same conditions as the experimental PCR tubes.

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The micropipettor should be set to about half the reaction volume of the master mix when mixing, and care should be taken to avoid introducing bubbles. Put caps on the 0. Once the lid to the thermal cycler is firmly closed start the program see Table 2. When the program has finished, the 0. PCR products can be detected by loading aliquots of each reaction into wells of an agarose gel then staining DNA that has migrated into the gel following electrophoresis with ethidium bromide. If a PCR product is present, the ethidium bromide will intercalate between the bases of the DNA strands, allowing bands to be visualized with a UV illuminator.

When setting up multiple PCR experiments, it is advantageous to assemble a mixture of reagents common to all reactions i.

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For instance, if there are 10 x 0. The reagents in the Master Mix are mixed thoroughly by gently pumping the plunger of a micropipettor up and down about 20 times as described above. Each PCR tube receives an aliquot of the Master Mix to which the DNA template, any required primers, and experiment-specific reagents are then added see Tables 1 and 7. False positives may occur as a consequence of carry-over from another PCR reaction which would be visualized as multiple undesired products on an agarose gel after electrophoresis.

Therefore, it is prudent to use proper technique, include a negative control and positive control when possible. While ethidium bromide is the most common stain for nucleic acids there are several safer and less toxic alternatives. While most modern PCR machines use 0. See your thermal cyclers manual to determine the appropriate size tube. Knowing the melting temperature T m of the primers is imperative for a successful PCR experiment.

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Although there are several T m calculators available, it is important to note that these calculations are an estimate of the actual T m due to lack of specific information about a particular reaction and assumptions made in the algorithms for the T m calculators themselves. The former will give more accurate T m estimation because it takes into account the stacking energy of neighboring base pairs.

The latter is used more frequently because the calculations are simple and can be done quickly by hand.

See Troubleshooting section for information about how various PCR conditions and additives affect melting temperature. PCR thermal cyclers rapidly heat and cool the reaction mixture, allowing for heat-induced denaturation of duplex DNA strand separation , annealing of primers to the plus and minus strands of the DNA template, and elongation of the PCR product.

Any longer than 3 minutes may inactivate the DNA polymerase, destroying its enzymatic activity. One method, known as hot-start PCR, drastically extends the initial denaturation time from 3 minutes up to 9 minutes. This protocol modification avoids likely inactivation of the DNA polymerase enzyme. Refer to the Troubleshooting section of this protocol for more information about hot start PCR and other alternative methods.

The next step is to set the thermal cycler to initiate the first of 25 to 35 rounds of a three-step temperature cycle Table 2. While increasing the number of cycles above 35 will result in a greater quantity of PCR products, too many rounds often results in the enrichment of undesirable secondary products. The three temperature steps in a single cycle accomplish three tasks: the first step denatures the template and in later cycles, the amplicons as well , the second step allows optimal annealing of primers, and the third step permits the DNA polymerase to bind to the DNA template and synthesize the PCR product.

The duration and temperature of each step within a cycle may be altered to optimize production of the desired amplicon. The time for the denaturation step is kept as short as possible.

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Usually 10 to 60 seconds is sufficient for most DNA templates. The denaturation time and temperature may vary depending on the G-C content of the template DNA, as well as the ramp rate, which is the time it takes the thermal cycler to change from one temperature to the next.

The temperature for this step is usually the same as that used for the initial denaturation phase step 1 above; e.

The cycle concludes with an elongation step. The temperature depends on the DNA polymerase selected for the experiment. Pfu DNA Polymerase is recommended for use in PCR and primer extension reactions that require high fidelity and requires 2 minutes for every 1 kb to be amplified.

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See manufacturer recommendations for exact elongation temperatures and elongation time indicated for each specific DNA polymerase. The final phase of thermal cycling incorporates an extended elongation period of 5 minutes or longer. This last step allows synthesis of many uncompleted amplicons to finish and, in the case of Taq DNA polymerase, permits the addition of an adenine residue to the 3' ends of all PCR products.

This modification is mediated by the terminal transferase activity of Taq DNA polymerase and is useful for subsequent molecular cloning procedures that require a 3'-overhang. The stringency of a reaction may be modulated such that the specificity is adjusted by altering variables e. For example, if the reaction is not stringent enough, many spurious amplicons will be generated with variable lengths. If the reaction is too stringent, no product will be produced.