Primer is a sequence that stimulates the synthesis of another macromolecule at the onset of nucleotide polymerization and is covalently bonded to the reactant. In nucleic acid chemistry, a primer is a short single-stranded piece of RNA or DNA fragment that binds to a region of the nucleic acid chain that is complementary to it and functions as the initiation point for nucleotide polymerization, from which nucleic acid polymerase can start synthesizing a new nucleic acid chain at its 3′ ends. Whether you do normal PCR or fluorescent quantitative PCR, designing the appropriate primers is a very critical step.

The detection methods of normal PCR and fluorescence quantitative PCR have major differences. Fluorescent quantitative PCR monitors the fluorescence excited by a fluorescent dye bound to DNA in real-time. Normal PCR determines the amount of PCR end product by detecting the amount of nucleic acid dye inserted into the DNA. Fluorescent quantitative PCR allows precise quantification by means of amplification curves, while ordinary PCR is qualitative by means of electrophoresis. So what are the similarities and differences in primer design between normal PCR and fluorescent quantitative PCR?

1. Similarities

• The sequence finding is the same.
• Sequence selection should be in the conserved region of the gene.
• Select the appropriate amplification fragment size.
• Avoid the formation of 4 or more consecutive pairs between primers themselves or with primers.
• Avoid primers forming a loop hairpin structure by themselves.
• Tm value at 55-65°C and GC content at 40%-60%.
• Avoid Tm differences between primers of more than 2°C.
• Avoid 3 or more consecutive identical bases at the 3' end of the primers.

2. Different places

2.1 Fluorescent quantitative PCR primers

• PCR product length: within 300bp is required, generally between 80-150bp is preferred.
• Primer specificity: high requirement for primers, minimize the generation of the primer dimer, melting curve requires single product.
• Amplification efficiency: The purpose of fluorescent quantitative PCR is to perform quantitation or relative quantification, and the amplification efficiency should be between 90% and 110%.
• For simultaneous amplification of multiple pairs of target genes, the primer conditions found in the literature will be different, and it is necessary to design primers with the most consistent conditions possible.
• When the target gene content is relatively low, primers with high sensitivity need to be designed.

2.2 Common PCR primers

• The amplification length of normal PCR generally ranges from 150 bp to several thousand bp, depending on the requirements of the experiment.
• Compared with fluorescence PCR, normal PCR requires less secondary structure.
• The requirement for amplification efficiency is not high.
• A gradient PCR instrument can be used to select different annealing temperatures, and the requirements for primer annealing temperatures do not need to be consistent.