PCR for Sanger Sequencing

PCR is used amplify the DNA region of interest prior to Sanger sequencing. The PCR reaction consists of the 5 components described below.

To quickly and easily obtain PCR primers, select and order predesigned PCR and Sanger sequencing primer pairs with our Invitrogen™ Primer Designer Tool, an online collection of ~650,000 primer pairs targeting the human exome and human mitochondrial genome

Primer design software, such as OligoPerfect software, 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.

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)

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

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).

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 Applied Biosystems™ 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.

The success or failure of most Applied Biosystems™ 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 4), 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%.

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.