Biomedical Engineering Reference
In-Depth Information
sequences, for determining primer location, orientation, and
efficiency of binding, and for calculating primer
T
m
and annealing
temperature in PCR.
The user must input a preexisting primer list into a second
Additional sequence
(
s
)
or pre
-
designed primers
(
probes
)
list
text editor. The number of preexisting primers is not limited; it can
be as many as the user needs. The target sequences can be entered
either as multiple separate DNA sequences or by opening files from
the selected folders. For in silico PCR against whole genome(s) or
a list of chromosomes, the user must specify the directory contain-
ing the input. The program will be consistent: it will look at each
file to find the position of the primers. The user can execute the
search task with
F5
on the
in silico
PCR
tab or can specify search
options including stringency and PCR product detection settings.
For the stringency options, users can specify the number of mis-
matches that the primers are allowed at 3′ terminus. The default
specificity settings allow a maximum two mismatches within the 3′
end region of the primers. These mismatches within the 3′ end of
the primers should not be located close to each other. Once the
primer set design is complete, the results will appear in the text
editors
In silico
PCR Result
.
In silico
PCR Result
text editor reports the specificity of the
primers (locations, including target position, similarity, and
T
m
), a
summary of primer pairs in relation to the PCR template, and
detailed information on each primer pair, including its length and
T
a
. It will show the target-specific primers that have been found.
The actual targets will be listed along with detailed alignments
between primers and targets (Fig.
8
).
6
Primer Analyses
Individual and sets of primers are evaluated using FastPCR or the
online software. They calculate primer
T
m
s using default or other
formulae for features of the primers including normal and degen-
erate nucleotide combinations, CG content, extinction coeffi-
cient, unit conversion (nmol per OD), mass (μg per OD),
molecular weight, and linguistic complexity and consider primer
PCR efficiency. Users can select either DNA or RNA primers
(online: PrimerAnalyser,
http://primerdigital.com/tools/
PrimerAnalyser.html
) with normal or degenerate oligonucleotides
or modifications with various labels (for example, inosine, uridine,
or fluorescent dyes). Tools allow the choice of other nearest
neighbor thermodynamic parameters or non-thermodynamic
T
m
calculation formulae.
For LNA modifications the four symbols, dA = E, dC = F,
dG = J, and dT = L, are used. Both programs perform analyses on-
