Using Web Primer: PCR
Location of Primers (with respect to a chosen Locus)
When the DNA source is a Locus, the location of the primers will be
determined relative to the start and stop codons of the gene. The
default option will find "forward" primers of given length(s) that
wholly reside somewhere within the first 35 basepairs upstream of the coding
sequence, and likewise will find "reverse"
primers that reside within the 35 basepairs immediatyely following the coding
sequence. The user may alter the endpoints
of either of these "primer selection regions" by changing the number
in the "Distance from Start" and "Distance from Stop" fields (note
that entering negative numbers will cause the primer selection region
to be located inside the coding region of the gene). The user may also define exact 5' endpoints of the
primers by selecting the button marked "YES" on the line which asks
about exact endpoints. Thus while the default option will allow
amplification of a region whose endpoints are somewhere within 35
basepairs upstream and 35 bp downstream of the gene, choosing an exact
endpoint will cause each primer to share the same 5' end.
Location of primers (with respect to DNA entered)
Primer location is most influenced by selection of a gene. Possible primers are determined by
their relationship to this gene. The user may choose where in relation to the start and stop
codons the primers are located. The default option will find primers in the first and last 35
basepairs of the DNA sequence entered. The user may define exact endpoints to start and stop the
primer, thus the default option will allow amplification of a region whose endpoints are in
the first and last 35 basepairs, while choosing exact enpoints will cause all primers evaluated to
start and end with exactly the same sequence.
Primer composition
Primers that contain a skewed AT/GC ratio can fail to give high
specificity, or yield primers that are in other ways not well
behaved. The user is allowed to enter minimum, optimal, and maximum
values for the percentage of basepairs which are either G or C.
Primer melting temperature
Melting
temperature heavily influence the results of PCR. This utility
calculates the Tm of an oligonucleotide by the nearest neighbor
method. (For more information, see Borer et al. 1974,
Rychlik et al. 1990, or Breslauer et
al. 1986.) Minimum, optimum, and maximum values may be set by the
user.
Primer Annealing
Primers also tend to
dimerize and anneal to themselves, this can present significant
problems in using PCR. One method for accounting for this problem was
developed by Hillier
and Green 1991 and calculates a local alignment score, either
anywhere within the primer or anchored from the 3'-end. For a visual
representation of this problem, see the help
from Primer3 on max complementarity and max 3' complementarity.
Maximum allowable alignment scores for annealing between primers can
be set by the user.
Ranking the pairs of primers
Primers are assigned a value based on their features and based on the
user defined preferences. The best pair of primers is defined to be
the pair of primers with the lowest score. This score is calculated
in the following way: +1 per 10% difference from the optimal GC
percentage, +1 per degree Celsius difference from the optimal Tm, +1
per 5 units of annealing at the end of primers, +1 per 10 units of
annealing in the middle of primers, and +1 per 2 basepairs difference
from the optimal length.
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