John M. S. Bartlett.pdf - Bio-Nica.info

84 Hyndman and Mitsuhashi
Fig. 1. Hairpin structures.
the polymerase and thereby taken out of the equilibrium between bound and unbound
templates. Because the primer concentration will essentially be unchanged, half of the
remaining templates will then be bound by primers and extended. In this way, most
templates can be extended if the extension is done at the T m of the primer.
If, however, the T m of the primer is a few degrees below the extension temperature,
only a small percentage of the primers will be hybridized, and the PCR will not be
efficient. Therefore, very efficient and specific PCR can be performed with two-step
cycling, but a sufficiently high primer T m is very important.
3.1.6. Hairpins
A hairpin is a structure formed by a single DNA molecule in which a portion on
one part of the DNA hybridizes to a complementary portion within the same DNA
strand, forming a structure resembling a hairpin (Fig. 1A). When a PCR primer forms
a hairpin, it adversely affects the primer’s ability to bind and extend at the target site.
In the worst case, the hairpin includes a base pair of the 3′-end and an overhang of the
5′-end (Fig. 1B). Such a structure allows the extension by DNA polymerase along the
primer and will result in the formation of a primer that will not be complementary to
the template and will not be extended if hybridized (Fig. 1C). In addition to removing
primers from the mixture, this also will prevent native primers from binding as target
sites that are bound by the extended primers. To avoid this, primers should be selected
that do not have any possible hairpin structures if possible.
3.1.7. Primer-Dimer Formation
The hybridization of two primers together is referred to as a primer-dimer (Fig. 2A).
There are two possibilities for these, homodimers and heterodimers. Homodimers are
formed from the hybridization of the same species of primer together. Heterodimers
are the duplex of two different primer sequences hybridizing together. The result of
either of these is that the primers will not be as efficient in hybridizing to the target.