Optimal design of the PCR primers is essential for accurate and specific quantification using real-time PCR. For the assay described herein, detection is based on the binding of the SYBR® Green I dye into double-stranded PCR products, which is a sequence-independent process. While this assay is cheaper than the specific probe-based assays, it loses the additional level of specificity introduced by the hybridization of a specific fluorescent TaqMan® probe to the PCR product. The sensitivity of detection with SYBR® Green may therefore be compromised by the lack of specificity of the primers, primer concentration (which can be limiting) and the formation of secondary structures in the PCR product. The formation of primer-dimers may register false positive fluorescence, however, this can now easily be overcome by running a PCR melting curve analysis. The 5' nuclease assay using TaqMan® probes would also be compromised by the lack of primer specificity and limiting primer concentration, and although these are not detected by the TaqMan® probe, they alter the amplification efficiency of the PCR reaction.
Primer design for SYBR® Green based assays needs to be more stringent than for a classic TaqMan® assay. It largely depends on the sequence targeted, and varies with the type of assay required: gene amplification, rearrangement, deletion, cDNA quantification, splicing variant and others. Primer design is still very restrictive: the annealing temperature is limited to between 58 and 60°C, which correspond to the optimal working conditions for the Taq DNA polymerase enzyme, and the length of the PCR product has to be set between 80 and 150 bp. The use of software dedicated to realtime PCR primer design, such as Primer Express® (Applied Biosystems), is highly recommended. However, it has rarely proven efficient using the TaqMan® primer/probe design option and is rather more successful using the simple DNA PCR option.
The first step in primer design is to determine the sequence that needs to be targeted by the PCR. This is highly dependent on the type of assay (gene amplification, rearrangement, deletion, gene expression, splicing variant, promoter switch, chromatin immunoprecipitation or others), which restricts the sequence available to design primers. The second step is to identify related genes and eliminate regions of high homology, which may further restrict the length of the sequence available. A third step in primer design for cDNA quantification requires the design to overlap exon-exon boundaries.
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