Figure 1. Reverse-primer tailing chemistry improves allele calling. (A) Illustration of two individuals with the same genotype analyzed for the same dinucleotide repeat marker using untailed primer. Di-, tri-, and tetranucleotide repeats tend to generate complex patterns due to the combination of stutter and the plus A artifact. In this example, the stutter peaks are 214 and 212, and the plus A artifacts are peaks 217, 215, and 213. Peak 216 is the correct allele peak for both samples. GeneMapper® software might not correctly call the alleles even though the pattern is visible for both offspring because the allele peak (216) is the highest peak for offspring 1, whereas the allele plus A peak (217) is the highest peak for offspring 2. Data of this type require manual editing to avoid missed or incorrect allele calls when reverse-primer tailing chemistry is not employed. (B) In this example, the 106 peak is the allele peak in the untailed product. The 114 peak is the allele peak plus A in the tailed product, which is 8 bases longer because it includes the 7-base tail and the additional A. Since GeneMapper® Software filters out stutter peaks automatically, elimination of the plus A problem results in easily called alleles.
Fragment Analysis: Setting Up the PCR Reaction
The success or failure of most GeneScan® 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 for you to analyze many independent loci in a single capillary injection. To maximize the amount of data you collect 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 fragments are labeled with: | Choose a size standards labeled with: | Recommended Size Standard | Possible Applications | Other Kits and Products | ||
|---|---|---|---|---|---|---|
| dR110, dR6G, dTAMRA™, dROX (Dye Set DS-02 - Filter E5) | LIZ® |
| ||||
| 5-FAM™, HEX™, NED™ (Dye Set DS-30 - Filter D) | ROX™ | GeneScan™ 500 ROX™ Size Standard | Custom Fragment Analysis | Custom Labeled Primer | ||
| 6-FAM™, VIC®, NED™ (Dye Set DS-31 - Filter D**) | ROX™ | GeneScan™ 500 ROX™ Size Standard | Custom Fragment Analysis | Custom Labeled Primer | ||
| 5-FAM™, JOE, NED™ (Dye Set DS-32 - Filter F) | ROX™ | GeneScan™ 500 ROX™ Size Standard |
| |||
| 6-FAM™, VIC®, NED™, PET® (Dye Set DS-33 - Filter G5) | LIZ® | GeneScan™ 600 LIZ Size Standard v2.0 |
|
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%.
PCR Component 3: MgCl2
MgCl2 (a co-factor of the AmpliTaq polymerase, absolutely necessary for a good enzyme activity.MgCl2 is chelated by the dNTP so increase of dNTP concentration require an increase of the MgCl2 concentration.
PCR Component 4: Buffer
This 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.
Multiplexing Strategies
A two-parameter approach (fluorescence label and fragment size) makes it possible for you 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 paris 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 the genetic analyzer.