Primer design is the most important factor in determining the success or failure of PCR. Here are some guidelines for designing and using primers in your PCR.
If you are performing seamless PCR cloning with our In-Fusion Cloning products, here are some specific primer design tips for this application.
What parameters do I need to consider when designing primers?
Primer design is the most important factor in determining the success or failure of PCR reactions. There are two major considerations for primer design: specificity and efficiency.
Specificity is determined by the frequency of mispriming events. Primers with poor specificity tend to produce undesired amplicons.
Efficiency is defined as the ability of primers to amplify a product with a two-fold increase per cycle to the theoretical optimum.
The following tables provide guidelines for primer design.
Guidelines
Length
The optimal length of primers is about 24 or 25 bases. However, length can be between 21 and 28 bases if the melting temperature (Tm) needs to be adjusted. When amplifying long DNA fragments (≥10 kb), 25- to 35-mer primers may provide better results.
GC content
The GC content (the number of Gs and Cs in the primer as a proportion of the total number of bases) should be 40–60%.
3' end
Having four G and/or C bases at the 3' end might be useful if the primer length is short (the bases provide "a clamping effect"), especially for universal primers, which are typically used for amplifying all cDNA or gDNA in a sample. However, adding these bases may increase non-specific priming events for gene-specific primers.
Tm
If possible, design primers with a melting temperature of 68–70°C. While this is not absolutely necessary, using stringent PCR conditions (e.g., "touchdown PCR" and "two-step PCR") can enhance primer specificity.
Sequence specificity
Primers should be specific to your gene of interest. A BLAST search can be used to find regions of homology. Note: PCR yield often depends on the 3' hexamer of the PCR primer. Primers that form a strong stable duplex actually reduce the amplification efficiency.
Avoid
Repeats
The maximum number of di-nucleotide repeats in a primer is four (e.g., ATATATAT).
Runs
Avoid long runs of a single base (more than three) as this can cause primer slippage and contribute to mispriming.
Complementary 3' ends
Forward and reverse primers should not anneal to each other and so should not have complementary G or C stretches (>4 contiguous bases).
Self-complementary 3' ends
Self-complementarity (e.g., within the forward primer) can lead to hairpin formation. A hairpin structure can form with just four G/C base pairs in the stem and three bases in the loop.
How do I calculate the melting temperature (Tm) of primers?
The primer melting temperature (Tm) is the estimate of DNA-DNA hybrid stability. Knowing the Tm is critical for determining an appropriate annealing temperature (Ta). A Ta that is too high will result in insufficient primer-template hybridization, leading to low PCR product yield. A Ta that is too low may lead to non-specific product amplification.
Calculation of the Tm of primers shorter than 20 bases can be performed using the Wallace rule:
Tm = 2°C (A+T) + 4°C (G+C)
For accurate estimation of the Tm of primers longer than 20 bases, we recommend using free primer design software such as Primer3.
What primer concentration should be used for PCR?
The final concentration of each primer should be between 0.1 and 0.5 µM. A stock solution of each primer is typically 10–20 µM.
For PCR amplicons less than 10 kb, 0.2 µM produces satisfactory results.
Primer concentrations that are too high increase the chance of mispriming, which may result in nonspecific amplification. Primer concentrations that are limiting can result in extremely inefficient amplification.
How should oligos be purified for PCR?
Standard desalted primers are satisfactory for most PCR applications.
What precautions should be taken when using inosine-containing primers?
Inosine-containing primers should not be used with PCR enzymes that have 3'→5' exonuclease activity (e.g., PrimeSTAR HS DNA Polymerase, PrimeSTAR Max DNA Polymerase, PrimeSTAR GXL DNA Polymerase, TaKaRa Ex Taq DNA Polymerase, or Takara LA Taq DNA polymerases), nor with Terra PCR Direct polymerase. When using one of the compatible TakaraTaqPCR enzymes for degenerate PCR, we recommend using a mixture of degenerate primers with A, T, G, or C at the desired position(s) rather than inosine-containing primers.
