primers extraction This product includes software developed by the
Whitehead Institute for Biomedical Research.
This tools outputs all repeat sequences (surrounded by 300 characters of flanking sequences) and prepares a primer3 input file. It then runs primer3 software on the input file and lets the user download the output file. Primer3 input options are described below:
PRIMER_EXPLAIN_FLAG (boolean, default 0)
If this flag is non-0, produce PRIMER_LEFT_EXPLAIN,
PRIMER_RIGHT_EXPLAIN, and PRIMER_INTERNAL_OLIGO_EXPLAIN output
tags, which are intended to provide information on the number of
oligos and primer pairs that Primer3 examined, and statistics on
the number discarded for various reasons. If -format_output is
set similar information is produced in the user-oriented output.
PRIMER_PICK_INTERNAL_OLIGO (boolean, default 0)
If the associated value is non-0, then Primer3 will attempt to
pick an internal oligo (hybridization probe to detect the PCR
product). This tag is maintained for backward compatibility.
PRIMER_GC_CLAMP (int, default 0)
Require the specified number of consecutive Gs and Cs at the 3'
end of both the left and right primer. (This parameter has no
effect on the internal oligo if one is requested.)
Optimum (default 20)
Optimum length (in bases) of a primer oligo. Primer3 will attempt
to pick primers close to this length.
PRIMER_MIN_SIZE (default 18)
Minimum acceptable length of a primer. Must be greater than 0
and less than or equal to PRIMER_MAX_SIZE.
PRIMER_MAX_SIZE (default 27)
Maximum acceptable length (in bases) of a primer. Currently this
parameter cannot be larger than 35. This limit is governed by
maximum oligo size for which Primer3's melting-temperature is
Primer melt Temp
Optimum (default 60.0C)
Optimum melting temperature(Celsius) for a primer oligo. Primer3
will try to pick primers with melting temperatures are close to
this temperature. The oligo melting temperature formula in
Primer3 is that given in Rychlik, Spencer and Rhoads, Nucleic
Acids Research, vol 18, num 12, pp 6409-6412 and Breslauer,
Frank, Bloeker and Marky, Proc. Natl. Acad. Sci. USA, vol 83, pp
3746-3750. Please refer to the former paper for background
Minimum (default 57.0C)
Minimum acceptable melting temperature(Celsius) for a primer
Maximum (default 63.0C)
Maximum acceptable melting temperature(Celsius) for a primer
Primer GC % content
Minimum (default 20.0%)
Minimum allowable percentage of Gs and Cs in any primer.
Optimum (default 50.0%)
Optimum GC percent. This parameter influences primer selection only if
PRIMER_WT_GC_PERCENT_GT or PRIMER_WT_GC_PERCENT_LT are non-0.
Maximum (default 80.0%)
Maximum allowable percentage of Gs and Cs in any primer generated
PRIMER_SELF_ANY (decimal,9999.99, default 8.00)
The maximum allowable local alignment score when testing a single
primer for (local) self-complementarity and the maximum allowable
local alignment score when testing for complementarity between
left and right primers. Local self-complementarity is taken to
predict the tendency of primers to anneal to each other without
necessarily causing self-priming in the PCR. The scoring system
gives 1.00 for complementary bases, -0.25 for a match of any base
(or N) with an N, -1.00 for a mismatch, and -2.00 for a gap.
Only single-base-pair gaps are allowed. For example, the
5' ATCGNA 3'
3' TA-CGT 5'
is allowed (and yields a score of 1.75), but the alignment
5' ATCCGNA 3'
|| | |
3' TA--CGT 5'
is not considered. Scores are non-negative, and a score of 0.00
indicates that there is no reasonable local alignment between two
PRIMER_SELF_END (decimal 9999.99, default 3.00)
The maximum allowable 3'-anchored global alignment score when
testing a single primer for self-complementarity, and the maximum
allowable 3'-anchored global alignment score when testing for
complementarity between left and right primers. The 3'-anchored
global alignment score is taken to predict the likelihood of
PCR-priming primer-dimers, for example
5' ATGCCCTAGCTTCCGGATG 3'
3' AAGTCCTACATTTAGCCTAGT 5'
5` AGGCTATGGGCCTCGCGA 3'
3' AGCGCTCCGGGTATCGGA 5'
The scoring system is as for the Maximum Complementarity
argument. In the examples above the scores are 7.00 and 6.00
respectively. Scores are non-negative, and a score of 0.00
indicates that there is no reasonable 3'-anchored global
alignment between two oligos. In order to estimate 3'-anchored
global alignments for candidate primers and primer pairs, Primer
assumes that the sequence from which to choose primers is
presented 5'->3'. It is nonsensical to provide a larger value
for this parameter than for the Maximum (local) Complementarity
parameter because the score of a local alignment will always be at
least as great as the score of a global alignment.
Max #N's (int, default 0)
Maximum number of unknown bases (N) allowable in any primer.