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

PCR Primer Design 87
(10,11). The entire statistical frequency of the entire oligonucleotide is calculated
based on the constituent 6-mers by starting with the 5′ terminal 6-mer frequency and
multiplying the relative frequency of the 5-mer on the 3′-end of that 6 mer having the
next nucleotide. This is repeated until the end of the oligonucleotide is reached.
For example, to calculate the frequency (f) of the 8-mer: CATAGCCT
f(CATAGCCT) =
4 f(ATAGCC)
f(CATAGC) × ———————————————————————
f(ATAGCT) + f(ATAGCG) + f(ATAGCC) + f(ATAGCT)
f(CATAGC)
4 f(TAGCCT)
f(CATAGC) × ———————————————————————
f(TAGCCA) + f(TAGCCG) + f(TAGCCC) + f(TAGCCT)
where f(CATAGC) denotes the frequency of the 6-mer, CATAGC.
3.2.4. 3′-Terminal Effects
Partial hybridization of the primer at the 3′-terminus can permit extension by DNA
polymerase. This could result in depletion of primers as well as possible nonspecific
amplification; therefore, this type of partial hybridization should be minimized as
much as possible.
There are two considerations for decreasing the chance of partial hybridization of
the 3′-terminal: frequency and stability.
3.2.5. 3′-Terminal Frequency
If the 3′-terminal region has a sequence that has many occurrences in the DNA
that will be in the reaction, then the likelihood of partial hybridization is greater. To
minimize this, the primers can be selected such that the 3′-terminal region does not
have a high frequency of occurrence within the genome of interest.
3.2.6. 3′-Terminal Stability
If the 3′-terminal region has a strong hybridization energy, then 3′-terminal partial
hybridizations will be relatively more stable. More stable 3′-terminal hybridization will
allow more false priming. Therefore if the primers are chosen such that the 3′-terminal
has a low hybridization strength (also referred to as terminal stability), the primer is
less likely to be priming as a result of such partial hybridization.
Note that in the efficiency section above, a stronger 3′-terminal stability is said
to improve efficiency. Whether one should select primers with high or low terminal
stability would depend on factors, such as the nature of the experiment (i.e., whether
there will be a large amount of other DNA) and the nature of the gene (whether highly
specific primers can be found).
3.2.7. Specificity Within the Target Sequence
If the primer is able to partially hybridize to a nonspecific region of the template,
particularly undesirable effects can occur. If the nonspecific hybridization allows
extension along the template such that a PCR product can be formed in conjunction
with one of the primers binding at one of the actual binding sites, nonspecific amplicons