Quantitative or real-time PCR is used as a routine assay for the monitoring the relative changes in gene expression under different experimental conditions. Designing of primers as well as probes during QPCR is one of the most crucial factors that affect the quality and success of the assay. Several guidelines are applicable for the design of primers for QPCR: GC content of primers should be 35-65%; melting temperature of the primers should be within 60-68 °C; one should also avoid secondary structures, the repeats of Gs or Cs that are longer than 3 bases, and the formation of primer-dimers.
Key Areas Covered
Key Terms: Fluorescent Dyes, GC Content, Melting Temperature, Primers, Quantitative PCR (QPCR)
What is QPCR
QPCR is a type of PCR that allows the quantification of the product in real time. Fluorescent dyes can be used in the quantification of PCR products by labelling them in each step. Two methods of fluorescent labeling can be used in QPCR assays. They are the use of fluorescent dyes and the fluorescently-labeled probes. Fluorescent dyes bind to the PCR product while probes anneal with the PCR product to form a stable triplex DNA. The widely-used fluorescent dye in QPCCR is SYBR Green while the probes can be Taqman. The use of probes in the detection of PCR products during QPCR gives more accurate results and increases the sensitivity of the assay.
How to Design Primers for QPCR
The designing of primers for QPCR is crucial in increasing the reliability, accuracy as well as the sensitivity of the assay. Guidelines for the design of QPCR primers are described below.
- PCR product/Amplicon size – The size of the PCR product should be 50-210 base pairs in size.
- Primer length – The length of the primers should be 19-23 nucleotides.
- GC content – The GC content of primers should be 35-65%.
- Melting temperature (Tm) – The melting temperature of primers should be 60-68 °C. The annealing temperature for the assay is 5 °C lesser than the Tm of the primers.
- Exon-exon junction – When amplifying cDNA by QPCR, the primers should span exon-exon junction to avoid the amplification of contaminating DNA.
- Repeats and runs – Dinucleotide repeats ( TCTCTCTCTC) and repeated nucleotides (eg. TAAAAAAAGC) should be avoided.
- 3’ Complementarity – The complementary regions of the 3’ ends of forward and reverse primers should be avoided to prevent the formation of primer-dimers.
- 3’ Stability – G or C residues should be included at the 3’ end of the primer to increase the stability of the annealing.
- GC clamp – One or two GC clamps at the 5’ end of the primer increases the specificity of the annealing.
- Specificity – The specificity of the primers should be checked by BLAST
- SNPs – Primers should not contain any known SNP (single nucleotide polymorphism) variations
Primers should be designed in such a way to avoid the formation of primer-dimers in QPCR. It is critical when using fluorescent dyes for the detection of the PCR product since these dyes also bind with the primer-dimers to give false positive results.
QPCR is used in the detection and quantification of PCR products. The designing of primers is crucial in QPCR to increase the accuracy of the results. Thus it is important to carefully follow the guidelines in designing primers for QPCR.
1. “QPCR Assay Design and Optimization.” LSR | Bio-Rad, Available here.