Biomedical Engineering Reference
In-Depth Information
and repeat content. PQ values of 80 and higher allow for the rapid
choice of the best PCR primer pair combinations. No adverse
effects, due to the modification of the reaction buffer, chosen ther-
mostable polymerases, or variations in annealing temperature, have
been observed on the reproducibility of PCR amplification using
primers with high PQ.
4.5 Hairpin (Loop)
and Dimer Formation
Primer-dimers involving one or two sequences may occur in a
PCR. The FastPCR tool eliminates intra- and inter-oligonucleotide
reactions before generating a primer list and primer pair candidates.
It is very important for PCR efficiency that the production of stable
and inhibitory dimers is prevented, especially by avoiding
complementarity in the 3′ ends of primers from whence the
polymerase will extend. Stable primer-dimer formation is very
effective at inhibiting PCR because the dimers formed are amplified
efficiently and compete with the intended target.
Primer-dimer prediction is based on analysis of non-gapped
local alignments and the stability of both the 3′ end and the central
regions of the primers (Fig.
2
). Primers will be rejected when they
have the potential to form stable dimers, depending on the nucleo-
tide composition and with at least five bases at the 3′ end or seven
bases in the central region. Tools to calculate
T
m
for primer-dimers
with mismatches for pure, mixed, or modified (inosine, uridine, or
locked nucleic acid (LNA)) bases, using averaged nearest neighbor
thermodynamic parameters, are provided for DNA/DNA duplexes
[
12
-
14
,
23
,
24
].
In addition to Watson-Crick base-pairing, there is a variety of
other hydrogen bonding configurations possible [
19
,
25
-
27
],
including G/C-quadruplexes and wobble base pairs, which the
FastPCR software detects. The program provides for the detection
of alternative hydrogen bonding during primer-dimer and in silico
PCR primer binding site detection. The mismatch stability is exam-
ined in order of decreasing stability:
G
-
C
>
A- T
> G · G > G · T ≥
G · A > T · T ≥ A · A > T · C ≥ A · C ≥ C · C. Guanine is the most universal
base, because it forms the strongest base pair and the strongest
mismatches. However, cytosine is the most discriminating base,
because it forms the strongest pair and the three weakest mis-
matches [
23
,
28
]. Therefore, the software also looks for stable
guanine mismatches: G · G, G · T and G · A.
G-rich (and C-rich) nucleic acid sequences can fold into four-
stranded DNA structures that contain stacks of G-quartets [
19
].
These quadruplexes can be formed by the intermolecular associa-
tion of two or four DNA molecules, dimerization of sequences
that contain two G bases, or by the intermolecular folding of a
single strand containing four blocks of guanines. These are easy to
eliminate from primer design because of their low linguistic com-
plexity; LC = 32 % for (TTAGGG)
4
. The software predicts the pres-
ence of putative G- and C-quadruplexes in primer sequences.
