PCR setup 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.

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. To quickly and easily obtain PCR primers, select and order predesigned PCR and Sanger sequencing primer pairs with our Primer Designer™ Tool, an online collection of >300,000 primer pairs targeting the human exome.

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Figure 1. Sequencing with PCR primer and standard primer tail.

To design optimal PCR primers, follow these recommendations:

  • 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 melting temperature 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 should not exceed 3 Gs or Cs
  • 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)

Why Use M13 Tailed PCR Primer or Other Universal Tailed Primer?

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Figure 2. Sequencing with PCR primer and standard primer tail.

For most large projects, it has become customary to include a standard (universal) primer tail on the PCR primers to simplify sequencing setup (Figure 1). The most common tail is the M13 sequence because it was initially used for sequencing clones constructed in the single-stranded bacteriophage M13. Potential disadvantages of using tailed PCR primers are the greater challenge in designing primers with a tail and the need for higher-quality oligonucleotides due to the increase in primer length (PCR primers).Life Technologies provides great primer design tools and an attractive price for primers up to 45 bases long.

The main advantage of using an M13 tailed primer or universal tailed primer is the simplicity of the sequencing reaction setup (Figure 2).

PCR Component 2: DNA Polymerase

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Figure 3. 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 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.

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.

Fragment analysis: setting up the PCR reaction

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).

When DNA sample fragments are labeled with the following dyes Choose a size standards labeled with the following dye Applied Biosystems® dye set Possible applications Associated Applied Biosystems® kits and products
dR110, dR6G, dTAMRA™, dROX LIZ® DS-02 SNaPshot® kit SNaPshot® primer focus kits, SNaPshot® multiplex kits
5-FAM™, HEX™, NED™ ROX™ DS-30 Custom Oligos
6-FAM™, VIC®, NED™ ROX™ DS-31

Microsatellites

Custom Oligos
5-FAM™, JOE, NED™ ROX™ DS-32 Microsatellites (Forensic) AmpFlSTR® Profiler Plus®, COfiler®, SGM Plus® Kits, StockMarks® Kits, Plant/Microbial AFLP Kits
6-FAM™, VIC®, NED™, PET® LIZ® DS-33 Microsatellites (Forensic) AmpFISTR® Identifiler® Kits, AmpFISTR® Yfiler® Amplification Kits, Custom StockMarks® Kits
6-FAM™, TET, HEX™ TAMRA™ DS-34
6-FAM™, dR6G LIZ® DS-40 SNP genotyping SNPlex® Genotyping System


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%.

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Figure 4. 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, 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.