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PCR Primer Design 85
Fig. 2. Dimer structures.
As with hairpins, the worst case is that in which the 3′-end of one of the primers is
base paired and there is a 5′ overhang (Fig. 2B). In this case, the primer will extend,
using the other primer as a template, rendering the extended primer unable to prime
the desired template (Fig. 2C). Even worse than with hairpins, this situation leads to
amplification of the primer dimers and rapid depletion of useable primers. To prevent
this, primer pairs should be chosen such that primer-dimer formation is minimal.
3.1.8. 3′-Terminal Stability
3′-terminal stability can loosely be defined as the relative hybridization strength
of the 3′-end of the primer. If the 3′-end of the primer has a low stability, it may not
efficiently prime because of the transient fraying of the end of the duplex. Therefore, a
higher 3′-terminal stability will improve priming efficiency. As will be mentioned later,
however, this high stability can have an undesirable affect on specificity.
3.2. Specificity
Specificity can generally be defined as the tendency for a primer to hybridize to its
intended target and not to other, nonspecific, targets. There are a few ways in which
poor specificity can impair PCR. First, if primers are hybridizing to many locations
nonspecifically, they will not be available to prime the target sequence. Second, if such
nonspecific hybridization were to occur, priming could also occur at those nonspecific
locations, which would effectively remove the primers from the reaction permanently.
Finally, by priming nonspecifically, it can be possible to generate aberrant amplicons.
This will not only obfuscate an assay for successful PCR, but will very rapidly consume
the primers to remove them from the reaction for amplifying the intended target.
3.2.1. Specificity, T m , and PCR Conditions
With respect to the annealing and extension temperatures chosen for the PCR
reaction, there is a balance that must be reached between considerations of efficiency
and specificity. As discussed previously, a more efficient PCR will result from having
primer T m equal to or above the extension temperature of the reaction. However,
having a primer with a high T m can often result in poor specificity. In such
cases, partial hybridization of the primer may be likely and extension can occur from
nonspecific sites. Such issues are less critical if highly specific primers can be selected
as discussed here.