PCR Cleanup

The success or failure of most GeneScan™ size standard fragment analysis experiments depends upon the success or failure of the PCR amplification step.

PCR Component 1: Primer Pair

A PCR primer pair consists of two oligonucleotides, typically 15–30 nucleotides in length, that hybridize to complementary strands of the DNA template and flank the region of interest. One primer in the pair is labeled with a fluorescent dye, so the PCR product will be detectable during capillary electrophoresis (CE) on the genetic analysis instrument. This two-parameter approach (fluorescence label and fragment size) makes it possible to analyze many independent loci in a single capillary injection. To maximize the amount of data collected in a single CE run, use a combination of dyes that display in different colors and can be detected by the same virtual filter set (see table below).

Plus A Artifact

One artifact of PCR amplification is the “plus A” peak, which results from non templated A nucleotide additions. Plus A artifacts increase the complexity of the peak pattern, making it more difficult to recognize true allele peaks. Reaction conditions can greatly impact these locus-dependent artifacts. Plus A artifacts occur when the polymerase copying a DNA strand adds an additional base (plus A) at the end of the sequence. The percentage of plus A added (0–100%) depends on the last 7 bases of the PCR product. To analyze the result, the plus A peak must be higher than the allele peak. Ambiguity in allele calling can result when the allele and allele plus A peaks are of near equal height (Figure 1), which occurs for approximately 5–10% of markers.

The patented reverse-primer tailing chemistry of the Custom Tailed Primer Pair improves allele-calling efficiency by eliminating the problems associated with nontemplated nucleotide addition. Primer tailing is effective because it controls the sequence context at the point where the polymerase binds to the end of double-stranded DNA, adding the nontemplated nucleotide. The tailed reverse primer contains a sequence of 7 bases that generates plus A products at close to 100%.

Figure 1: Reverse-primer tailing chemistry improves allele calling. In this example, the 106 peak is the allele peak in the untailed product. GeneMapper® software might not correctly call the alleles because the allele peak and the allele plus A peak (107) are of similar heights. Very often, data of this type requires manual editing to avoid missed or incorrect allele calls when reverse-primer tailing chemistry is not employed. In contrast, in the tailed product, the 114 peak is the allele peak plus A, which is 8 bases longer because it includes the 7-base tail and the additional A. The untailed allele peak completely disappears, facilitating analysis by the software algorithm.

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 Gs or Cs
• The sequences should be analyzed to avoid complementarity and prevent hybridization between primers (primer-dimers)

Primer Design Software

Primer design software, such as OligoPerfect™ software (available at www.lifetech.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.

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 ensures that no active Taq is present during reaction setup and the initial DNA denaturation step. AmpliTaq® DNA Polymerase is a good choice.

PCR Component 3: MgCl2

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

PCR Component 4: Buffer

An optimized buffer is provided with the enzyme.

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.

A two-parameter approach (fluorescence label and fragment size) makes it possible to analyze many independent loci in a single capillary injection and greatly increases the instrument throughput. Two general multiplexing strategies include:

• Multiplex during the PCR reaction by combining more than one pair of primers in the same PCR reaction tube. This strategy significantly decreases the cost per analysis, but requires optimization. Multiplex primers must not produce products of similar lengths and cannot be labeled with the same fluorescent dyes. Primers cannot contain large regions of complementarity. Primers should have similar melting temperatures. Before performing the PCR using this strategy, perform a preliminary check for primer compatibility and test the pairs for successful co-amplification.
• Pooling PCR product after PCR. This strategy is simpler and more flexible, but pooling products from multiple PCR reactions often increases the salt concentration in the loaded samples, which can have unwanted downstream effects.

Note: PCR products used for fragment analysis don’t need to be purified before separation on a genetic analyzer.