Perhaps the most critical parameter for successful PCR is the design of Primers. All things being equal, a poorly designed primer can result in a PCR reaction that will not work. The primer sequence determines several things such as the length of the product, its melting temperature and ultimately the yield. A poorly designed primer can result in little or no product due to non-specific amplification and/or primer-dimer formation, which can become competitive enough to suppress product formation. This application note is provided to give rules that should be taken into account when designing primers for PCR. More comprehensive coverage of this subject can be found elsewhere(1).

Primer selection
Several variables must be taken into account when designing PCR Primers. Among the most critical are:
Primer length
Melting Temperature (Tm)
Specificity
Complementary Primer Sequences
G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches
3’-end Sequence

Each of these critical elements will be discussed in turn.
Primer length
Since both specificity and the temperature and time of annealing are at least partly dependent on primer length, this parameter is critical for successful PCR. In general, oligonucleotides between 18 and 24 bases are extremely sequence specific, provided that the annealing temperature is optimal. Primer length is also proportional to annealing efficiency: in general, the longer the primer, the more inefficient the annealing. With fewer templates primed at each step, this can result in a significant decrease in amplified product. The primers should not be too short, however, unless the application specifically calls for it. As discussed below, the goal should be to design a primer with an annealing temperature of at least 50 °C.
The relationship between annealing temperature and melting temperature is one of the “Black Boxes” of PCR. A general rule-of-thumb is to use an annealing temperature that is 5 °C lower than the melting temperature. Thus, when aiming for an annealing temperature of at least 50 °C, this corresponds to a primer with a calculated melting temperature(Tm) ~55 °C. Often, the annealing temperature determined in this fashion will not be optimal and empirical experiments will have to be performed to determine the optimal temperature. This is most easily accomplished using a gradient thermal cycler like Eppendorf's Mastercycler® gradient.

Melting Temperature (Tm)

It is important to keep in mind that there are two primers added to a PCR reaction. Both of the oligonucleotide primers should be designed such that they have similar melting temperatures. If primers are mismatched in terms of Tm, amplification will be less efficient or may not work at all since the primer with the higher Tm will mis-prime at lower temperatures and the primer with the lower Tm may not work at higher temperatures.

The melting temperatures of oligos are most accurately calculated using nearest neighbor thermodynamic calculations with the formula:

T_m^primer = ∆H [∆S+ R ln (c/4)] –273.15 °C + 16.6 log ₁₀ [K⁺]

where H is the enthalpy and S is the entropy for helix formation, R is the molar gas constant and c is the concentration of primer. This is most easily accomplished using any of a number of primer design software packages on the market⁽³⁾. Fortunately, a good working approximation of this value (generally valid for oligos in the 18–24 base range) can be calculated using the formula:

T_m = 2(A+T) + 4(G+C).

The table below shows calculated values for primers of various lengths using this equation, which is known as the Wallace formula, and assuming a 50% GC content⁽⁴⁾.

[1] [2] [3] 下一页