Introduction
Primer design and usage guidelines
Performing PCR primer design requires careful consideration and precise methodology to ensure the success of the PCR process. Optimal primer design is critical for achieving maximal specificity and efficiency, making it a cornerstone of accurate molecular biology experiments. "PCR primer design" involves selecting sequences that not only hybridize perfectly with the target DNA but also resist forming secondary structures or dimers that can hinder the PCR reaction.
Optimal primer sequences and appropriate primer concentrations are essential for maximal specificity and efficiency in PCR. The table, Primer design and usage guidelines, provides an overview of primer design and use for standard and multiplex PCR, as well as one-step RT-PCR. Molar conversions can be found in the table Molar conversions for PCR primers.
Primer design and usage guidelines
| Standard PCR | Multiplex PCR | One-step RT-PCR |
|
|---|---|---|---|
| Length | 18–30 nt | 21–30 nt | 18–30 nt |
| GC content | 40–60% | 40–60% | 40–60% |
| Tm information | The Tm of all primer pairs should be similar |
The Tm of all primer pairs should be similar. For optimal results, the Tm should be 60–88°C |
The Tm of all primer pairs should be similar. The Tm should not be lower than the temperature of the reverse transcription (e.g., 50°C) |
| Estimating optimal annealing temperature |
5°C below the calculated Tm |
5–8°C below the calculated Tm (when greater than 68°C) or 3–6°C below the calculated Tm (when 60–67°C) |
5°C below the calculated Tm |
| Location | – | – | To prevent detection of gDNA:Primer hybridizes to the 3' end of one exon and the 5' end of the adjacent exon. Alternatively, the primer hybridizes to a flanking region that contains at least one intron. If only the mRNA sequence is known, choose primer annealing sites that are 300–400 bp apart. |
| Concentration, A260 unit equivalence |
20–30 µg | 20–30 µg | 20–30 µg |
Molar conversions for PCR primers
| Primer length | pmol/µg | 20 pmol |
|---|---|---|
| 18mer | 168 | 119 ng |
| 20mer | 152 | 132 ng |
| 25mer | 121 | 165 ng |
| 30mer | 101 | 198 ng |
The following points should be considered when designing PCR primers and are common to all types of PCR:
- Tm calculation: 2°C x (A+T) + 4°C x (G+C)
- Avoid complementarity in the 2–3 bases at the 3' end of the primer pairs
- Avoid mismatches between the 3' end of the primer and the template
- Avoid runs of 3 or more Cs or Gs at the 3' end of the primer
- Avoid complementarity within primers and between the primer pair
- Avoid a T as ultimate base at the 3' end
- Ensure primer sequence is unique for the template sequence
- Use a concentration of 0.1–1.0 µM of each primer. For many applications, a primer concentration of 0.2 µM will be sufficient
Lyophilized primers should be dissolved in a small volume of distilled water or TE to make a concentrated stock solution. Prepare small aliquots of working solutions containing 10 pmol/µl to avoid repeated thawing and freezing. Store all primer solutions at –20°C. Primer quality can be checked on a denaturing polyacrylamide gel; a single band should be seen.