Sequencing: Setting Up the PCR Reaction

PCR Component 1: Primer Pair

A PCR primer pair consists of two oligonucleotides, typically 15–30 nucleotides long, which hybridize to complementary strands of the DNA template and flank the region of interest. Recommendations for good PCR primer design include:

  • Primers should be specific for the target sequence and be free of internal secondary structure
  • Primers should not include stretches of polybase sequences (e.g., poly (dG)) or repeating motifs, as these can hybridize inappropriately to the template
  • Primer pairs should have compatible melting temperatures (within 5°C) and contain approximately 50% GC content. High GC content results in the formation of stable imperfect hybrids, while high AT content depresses the Tm of perfectly matched hybrids. If possible, the 3´ end of the primer should be rich in GC bases (GC clamp) to enhance annealing of the end that will be extended, but not exceed 3 G or C
  • The sequences should be analyzed to avoid complementarity and prevent hybridization between primers (primer-dimers) (Figure 1)

Primer Design Software

Primer design software, such as OligoPerfect™ software (available at www.invitrogen.com/oligoperfect), can automatically evaluate a target sequence and design primers for it based on the criteria listed above. To confirm the specificity of your primers, a BLAST® search may be performed against public databases to be sure that your primers only recognize the target of interest.

Universal-Tailed Primers

You can synthesize a PCR primer that has a universal sequencing primer binding site added to the 5´ end. Universal-tailed PCR primers are useful in the following scenarios:

  • In conjunction with dye terminator chemistries (universal sequencing primers have good annealing characteristics)
  • In conjunction with commercially available dye-labeled sequencing primers
  • To sequence the resulting PCR product to simplify and standardize the sequencing step (this is the strategy behind the Applied Biosystems® BigDye® Direct Cycle Sequencing Kit)

PCR Component 2: DNA Polymerase

PCR performance is often related to the DNA polymerase, so enzyme selection is critical to success. One of the main factors affecting PCR specificity is the fact that Taq DNA polymerase has residual activity at low temperatures. Primers can anneal nonspecifically to DNA, allowing the polymerase to synthesize nonspecific product. This complication can be minimized by the inclusion of a hot-start enzyme. Using a hot-start enzyme helps ensure that no active Taq is present during reaction setup and the initial DNA denaturation step.

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Figure 1: Example of duplicated sequence caused by the formation of primer-dimers during the PCR reaction.

The primer-dimers anneal and are filled in to create short, nontemplate PCR products. These artifacts can be significantly reduced by ensuring your primers are designed well and that you incorporate a hot start enzyme.

PCR Component 3: MgCl2

MgCl2 a co-factor of AmpliTaq® polymerase, is critical for good enzyme activity. MgCl2 is chelated by dNTPs, so an increase in dNTP concentration requires an increase MgCl2 concentration.

PCR Component 4: Buffer

An optimized buffer is provided with the enzyme.

PCR Component 5: Additives

Some other additives may be incorporated to facilitate amplification of difficult templates (i.e., DMSO for GC-rich templates)

Performing the PCR Reaction

There are three major steps that make up a PCR reaction. Reactions are generally run for 30 cycles.

  1. Denaturation—the temperature should be appropriate to the polymerase chosen (usually 95°C). The denaturation time can be increased if template GC content is high.
  2. Annealing—use appropriate temperatures based on the calculated melting temperature (Tm) of the primers (5°C below the Tm of the primer).
  3. Extension—at 70–72°C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second.

PCR amplicons are typically evaluated using agarose gel electrophoresis. To obtain a good sequencing reaction, the PCR product should appear as a single band on an agarose gel. Multiple bands indicate sequence duplications (Figure 1).

PCR Cleanup

The goal of PCR cleanup is to remove the excess PCR primers (one primer is used in each sequencing reaction) and dNTPs (to preserve the ratio of the dNTP to ddNTP necessary for efficient BigDye® Cycle Sequencing reactions). There are several methods for purifying PCR products. Select a method based on the amounts of components carried over from the PCR reaction and on the sequencing chemistry you plan to use:

  • Ultrafiltration
  • Ethanol precipitation
  • Gel purification
  • Enzymatic purification: involves shrimp alkaline phosphatase (SAP) and Exonuclease I (Exo I) treatment before sequencing; the SAP/Exo I method degrades nucleotides and single-stranded DNA (primers) remaining after PCR

IMPORTANT! If more than one PCR product is present, column purification, ethanol precipitation, or enzymatic purification will not isolate the desired product. Use gel purification to isolate the desired product or reoptimize the PCR to obtain a single product. Ultrafiltration may work if the contaminating PCR products are much smaller than the desired PCR product.

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