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:
IMPORTANT! If more than one PCR product is present, column purification, ethanol precipitation, or enzymatic purification cannot be used to 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|| |
|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 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%.
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.
Performing the PCR Reaction
There are three major steps that make up a PCR reaction. Reactions are generally run for 30 cycles.
- 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.
- Annealing—use appropriate temperatures based on the calculated melting temperature (Tm) of the primers (5°C below the Tm of the primer).
- 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.