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<HTML>
<HEAD>
<TITLE>
EMBOSS: eprimer3
</TITLE>
</HEAD>
<BODY BGCOLOR="#FFFFFF" text="#000000">
<table align=center border=0 cellspacing=0 cellpadding=0>
<tr><td valign=top>
<A HREF="/" ONMOUSEOVER="self.status='Go to the EMBOSS home page';return true"><img border=0 src="emboss_icon.jpg" alt="" width=150 height=48></a>
</td>
<td align=left valign=middle>
<b><font size="+6">
eprimer3
</font></b>
</td></tr>
</table>
<br>
<p>
<H2>
Function
</H2>
Picks PCR primers and hybridization oligos
<H2>
Description
</H2>
<b>eprimer3</b> is an interface to the 'primer3' program from the
Whitehead Institute.
<p>
The Whitehead program must be set up and on the path in order for
<b>eprimer3</b> to find and run it.
<p>
Primer3 picks primers for PCR reactions, considering as criteria:
<p>
<UL>
<LI>oligonucleotide melting temperature, size, GC content,
and primer-dimer possibilities,
<LI>PCR product size,
<LI>positional constraints within the source sequence, and
<LI>miscellaneous other constraints.
</UL>
<p>
All of these criteria are user-specifiable as constraints.
<p>
<b>eprimer3</b> can also pick hybridisation oligos that are internal to
the product.
<H3>ADVICE FOR PICKING PRIMERS</H3>
We suggest referring to: Wojciech Rychlik, "Selection of Primers
for Polymerase Chain Reaction" in BA White, Ed., "Methods in
Molecular Biology, Vol. 15: PCR Protocols: Current Methods and
Applications", 1993, pp 31-40, Humana Press, Totowa NJ
<H4>Cautions</H4>
Some of the most important issues in primer picking can be
addressed only before using eprimer3. These are sequence quality
(including making sure the sequence is not vector and not
chimeric) and avoiding repetitive elements.
<p>
Techniques for avoiding problems include a thorough understanding
of possible vector contaminants and cloning artifacts coupled
with database searches using blast, fasta, or other similarity
searching program to screen for vector contaminants and possible
repeats. Repbase (J. Jurka, A.F.A. Smit, C. Pethiyagoda, and
others, 1995-1996, ftp://ncbi.nlm.nih.gov/repository/repbase)
is an excellent source of repeat sequences and pointers to the
literature. eprimer3 now allows you to screen candidate oligos
against a Mispriming Library (or a Mishyb Library in the case
of internal oligos).
<p>
Sequence quality can be controlled by manual trace viewing and
quality clipping or automatic quality clipping programs. Low-
quality bases should be changed to N's or can be made part of
Excluded Regions. The beginning of a sequencing read is often
problematic because of primer peaks, and the end of the read
often contains many low-quality or even meaningless called bases.
Therefore when picking primers from single-pass sequence it is
often best to use the INCLUDED_REGION parameter to ensure that
eprimer3 chooses primers in the high quality region of the read.
<p>
In addition, eprimer3 takes as input a Sequence Quality list for
use with those base calling programs
<p>
(e.g. Phred, Bass/Grace, Trout) that output this information.
<H4>What to do if eprimer3 cannot find a primers?</H4>
Try relaxing various parameters, including the
self-complementarity parameters and max and min oligo melting
temperatures. For example, for very A-T-rich regions you might
have to increase maximum primer size or decrease minimum melting
temperature. It is usually unwise to reduce the minimum primer
size if your template is complex (e.g. a mammalian genome), since
small primers are more likely to be non-specific. Make sure that
there are adequate stretches of non-Ns in the regions in which
you wish to pick primers. If necessary you can also allow an N
in your primer and use an oligo mixture containing all four bases
at that position.
<p>
Try setting the '-explain' option.
<H2>
Usage
</H2>
<b>Here is a sample session with eprimer3</b>
<p>
<p>
<table width="90%"><tr><td bgcolor="#CCFFFF"><pre>
% <b>eprimer3 tembl:x65921 x65921.eprimer3 -explain </b>
Picks PCR primers and hybridization oligos
</pre></td></tr></table><p>
<p>
<a href="#input.1">Go to the input files for this example</a><br><a href="#output.1">Go to the output files for this example</a><p><p>
<H2>
Command line arguments
</H2>
<table CELLSPACING=0 CELLPADDING=3 BGCOLOR="#f5f5ff" ><tr><td>
<pre>
Standard (Mandatory) qualifiers:
[-sequence] seqall The sequence from which to choose primers.
The sequence must be presented 5' to 3'
[-outfile] outfile [*.eprimer3] Whitehead primer3_core program
output file
Additional (Optional) qualifiers (* if not always prompted):
-[no]primer toggle [Y] Tell EPrimer3 to pick primer(s)
* -task menu [1] Tell EPrimer3 what task to perform.
Legal values are 1: 'Pick PCR primers', 2:
'Pick forward primer only', 3: 'Pick reverse
primer only', 4: 'No primers needed'.
(Values: 1 (Pick PCR primers); 2 (Pick
forward primer only); 3 (Pick reverse primer
only); 4 (No primers needed))
-hybridprobe toggle [N] An 'internal oligo' is intended to be
used as a hybridization probe (hyb probe) to
detect the PCR product after amplification.
* -oligomishyblibraryfile infile Similar to MISPRIMING-LIBRARY, except that
the event we seek to avoid is hybridization
of the internal oligo to sequences in this
library rather than priming from them.
The file must be in (a slightly restricted)
FASTA format (W. B. Pearson and D.J. Lipman,
PNAS 85:8 pp 2444-2448 [1988]); we briefly
discuss the organization of this file below.
If this parameter is specified then
EPrimer3 locally aligns each candidate oligo
against each library sequence and rejects
those primers for which the local alignment
score times a specified weight (see below)
exceeds INTERNAL-OLIGO-MAX-MISHYB. (The
maximum value of the weight is arbitrarily
set to 12.0.)
Each sequence entry in the FASTA-format file
must begin with an 'id line' that starts
with '>'. The contents of the id line is
'slightly restricted' in that EPrimer3
parses everything after any optional
asterisk ('*') as a floating point number to
use as the weight mentioned above. If the
id line contains no asterisk then the weight
defaults to 1.0. The alignment scoring
system used is the same as for calculating
complementarity among oligos (e.g.
SELF-ANY). The remainder of an entry
contains the sequence as lines following the
id line up until a line starting with '>'
or the end of the file. Whitespace and
newlines are ignored. Characters 'A', 'T',
'G', 'C', 'a', 't', 'g', 'c' are retained
and any other character is converted to 'N'
(with the consequence that any IUB / IUPAC
codes for ambiguous bases are converted to
'N'). There are no restrictions on line
length.
An empty value for this parameter indicates
that no library should be used.
-numreturn integer [5] The maximum number of primer pairs to
return. Primer pairs returned are sorted by
their 'quality', in other words by the value
of the objective function (where a lower
number indicates a better primer pair).
Caution: setting this parameter to a large
value will increase running time. (Integer 0
or more)
-includedregion range [(full sequence)] A sub-region of the given
sequence in which to pick primers. For
example, often the first dozen or so bases
of a sequence are vector, and should be
excluded from consideration. The value for
this parameter has the form
(start),(end)
where (start) is the index of the first base
to consider, and (end) is the last in the
primer-picking region.
-target range [(full sequence)] If one or more Targets is
specified then a legal primer pair must
flank at least one of them. A Target might
be a simple sequence repeat site (for
example a CA repeat) or a single-base-pair
polymorphism. The value should be a
space-separated list of
(start),(end)
pairs where (start) is the index of the
first base of a Target, and (end) is the
last
E.g. 50,51 requires primers to surround the
2 bases at positions 50 and 51.
-excludedregion range [(full sequence)] Primer oligos may not
overlap any region specified in this tag.
The associated value must be a
space-separated list of
(start),(end)
pairs where (start) is the index of the
first base of the excluded region, and and
(end) is the last. This tag is useful for
tasks such as excluding regions of low
sequence quality or for excluding regions
containing repetitive elements such as ALUs
or LINEs.
E.g. 401,407 68,70 forbids selection of
primers in the 7 bases starting at 401 and
the 3 bases at 68.
-forwardinput string The sequence of a forward primer to check
and around which to design reverse primers
and optional internal oligos. Must be a
substring of SEQUENCE. (Any string is
accepted)
-reverseinput string The sequence of a reverse primer to check
and around which to design forward primers
and optional internal oligos. Must be a
substring of the reverse strand of SEQUENCE.
(Any string is accepted)
* -gcclamp integer [0] Require the specified number of
consecutive Gs and Cs at the 3' end of both
the forward and reverse primer. (This
parameter has no effect on the internal
oligo if one is requested.) (Integer 0 or
more)
* -osize integer [20] Optimum length (in bases) of a primer
oligo. EPrimer3 will attempt to pick primers
close to this length. (Integer 0 or more)
* -minsize integer [18] Minimum acceptable length of a primer.
Must be greater than 0 and less than or
equal to MAX-SIZE. (Integer 1 or more)
* -maxsize integer [27] Maximum acceptable length (in bases) of
a primer. Currently this parameter cannot
be larger than 35. This limit is governed by
the maximum oligo size for which EPrimer3's
melting-temperature is valid. (Integer up
to 35)
* -otm float [60.0] Optimum melting temperature(Celsius)
for a primer oligo. EPrimer3 will try to
pick primers with melting temperatures are
close to this temperature. The oligo melting
temperature formula in EPrimer3 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 discussion. (Any
numeric value)
* -mintm float [57.0] Minimum acceptable melting
temperature(Celsius) for a primer oligo.
(Any numeric value)
* -maxtm float [63.0] Maximum acceptable melting
temperature(Celsius) for a primer oligo.
(Any numeric value)
* -maxdifftm float [100.0] Maximum acceptable (unsigned)
difference between the melting temperatures
of the forward and reverse primers. (Any
numeric value)
* -ogcpercent float [50.0] Primer optimum GC percent. (Any
numeric value)
* -mingc float [20.0] Minimum allowable percentage of Gs
and Cs in any primer. (Any numeric value)
* -maxgc float [80.0] Maximum allowable percentage of Gs
and Cs in any primer generated by Primer.
(Any numeric value)
* -saltconc float [50.0] The millimolar concentration of salt
(usually KCl) in the PCR. EPrimer3 uses this
argument to calculate oligo melting
temperatures. (Any numeric value)
* -dnaconc float [50.0] The nanomolar concentration of
annealing oligos in the PCR. EPrimer3 uses
this argument to calculate oligo melting
temperatures. The default (50nM) works well
with the standard protocol used at the
Whitehead/MIT Center for Genome
Research--0.5 microliters of 20 micromolar
concentration for each primer oligo in a 20
microliter reaction with 10 nanograms
template, 0.025 units/microliter Taq
polymerase in 0.1 mM each dNTP, 1.5mM MgCl2,
50mM KCl, 10mM Tris-HCL (pH 9.3) using 35
cycles with an annealing temperature of 56
degrees Celsius. This parameter corresponds
to 'c' in Rychlik, Spencer and Rhoads'
equation (ii) (Nucleic Acids Research, vol
18, num 12) where a suitable value (for a
lower initial concentration of template) is
'empirically determined'. The value of this
parameter is less than the actual
concentration of oligos in the reaction
because it is the concentration of annealing
oligos, which in turn depends on the amount
of template (including PCR product) in a
given cycle. This concentration increases a
great deal during a PCR; fortunately PCR
seems quite robust for a variety of oligo
melting temperatures.
See ADVICE FOR PICKING PRIMERS. (Any numeric
value)
* -maxpolyx integer [5] The maximum allowable length of a
mononucleotide repeat in a primer, for
example AAAAAA. (Integer 0 or more)
* -productosize integer [200] The optimum size for the PCR product.
0 indicates that there is no optimum product
size. (Integer 0 or more)
* -productsizerange range [100-300] The associated values specify the
lengths of the product that the user wants
the primers to create, and is a space
separated list of elements of the form
(x)-(y)
where an (x)-(y) pair is a legal range of
lengths for the product. For example, if one
wants PCR products to be between 100 to 150
bases (inclusive) then one would set this
parameter to 100-150. If one desires PCR
products in either the range from 100 to 150
bases or in the range from 200 to 250 bases
then one would set this parameter to
100-150 200-250.
EPrimer3 favors ranges to the left side of
the parameter string. EPrimer3 will return
legal primers pairs in the first range
regardless the value of the objective
function for these pairs. Only if there are
an insufficient number of primers in the
first range will EPrimer3 return primers in
a subsequent range.
* -productotm float [0.0] The optimum melting temperature for
the PCR product. 0 indicates that there is
no optimum temperature. (Any numeric value)
* -productmintm float [-1000000.0] The minimum allowed melting
temperature of the amplicon. Please see the
documentation on the maximum melting
temperature of the product for details. (Any
numeric value)
* -productmaxtm float [1000000.0] The maximum allowed melting
temperature of the amplicon. Product Tm is
calculated using the formula from Bolton and
McCarthy, PNAS 84:1390 (1962) as presented
in Sambrook, Fritsch and Maniatis, Molecular
Cloning, p 11.46 (1989, CSHL Press).
Tm = 81.5 + 16.6(log10([Na+])) + .41*(%GC) -
600/length
Where [Na+} is the molar sodium
concentration, (%GC) is the percent of Gs
and Cs in the sequence, and length is the
length of the sequence.
A similar formula is used by the prime
primer selection program in GCG
http://www.gcg.com), which instead uses
675.0/length in the last term (after F.
Baldino, Jr, M.-F. Chesselet, and M.E.
Lewis, Methods in Enzymology 168:766 (1989)
eqn (1) on page 766 without the mismatch and
formamide terms). The formulas here and in
Baldino et al. assume Na+ rather than K+.
According to J.G. Wetmur, Critical Reviews
in BioChem. and Mol. Bio. 26:227 (1991) 50
mM K+ should be equivalent in these formulae
to .2 M Na+. EPrimer3 uses the same salt
concentration value for calculating both the
primer melting temperature and the oligo
melting temperature. If you are planning to
use the PCR product for hybridization later
this behavior will not give you the Tm under
hybridization conditions. (Any numeric
value)
* -oligoexcludedregion range [(full sequence)] Middle oligos may not
overlap any region specified by this tag.
The associated value must be a
space-separated list of
(start),(end)
pairs, where (start) is the index of the
first base of an excluded region, and (end)
is the last. Often one would make Target
regions excluded regions for internal
oligos.
* -oligoinput string The sequence of an internal oligo to check
and around which to design forward and
reverse primers. Must be a substring of
SEQUENCE. (Any string is accepted)
* -oligoosize integer [20] Optimum length (in bases) of an
internal oligo. EPrimer3 will attempt to
pick primers close to this length. (Integer
0 or more)
* -oligominsize integer [18] Minimum acceptable length of an
internal oligo. Must be greater than 0 and
less than or equal to
INTERNAL-OLIGO-MAX-SIZE. (Integer 0 or more)
* -oligomaxsize integer [27] Maximum acceptable length (in bases) of
an internal oligo. Currently this parameter
cannot be larger than 35. This limit is
governed by maximum oligo size for which
EPrimer3's melting-temperature is valid.
(Integer up to 35)
* -oligootm float [60.0] Optimum melting temperature (Celsius)
for an internal oligo. EPrimer3 will try to
pick oligos with melting temperatures that
are close to this temperature. The oligo
melting temperature formula in EPrimer3 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 discussion. (Any
numeric value)
* -oligomintm float [57.0] Minimum acceptable melting
temperature(Celsius) for an internal oligo.
(Any numeric value)
* -oligomaxtm float [63.0] Maximum acceptable melting
temperature (Celsius) for an internal oligo.
(Any numeric value)
* -oligoogcpercent float [50.0] Internal oligo optimum GC percent.
(Any numeric value)
* -oligomingc float [20.0] Minimum allowable percentage of Gs
and Cs in an internal oligo. (Any numeric
value)
* -oligomaxgc float [80.0] Maximum allowable percentage of Gs
and Cs in any internal oligo generated by
Primer. (Any numeric value)
* -oligosaltconc float [50.0] The millimolar concentration of salt
(usually KCl) in the hybridization. EPrimer3
uses this argument to calculate internal
oligo melting temperatures. (Any numeric
value)
* -oligodnaconc float [50.0] The nanomolar concentration of
annealing internal oligo in the
hybridization. (Any numeric value)
* -oligoselfany float [12.00] The maximum allowable local
alignment score when testing an internal
oligo for (local) self-complementarity.
Local self-complementarity is taken to
predict the tendency of oligos to anneal to
themselves 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 alignment
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
oligos. (Number up to 9999.990)
* -oligoselfend float [12.00] The maximum allowable 3'-anchored
global alignment score when testing a single
oligo for self-complementarity.
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
oligos, Primer assumes that the sequence
from which to choose oligos is presented 5'
to 3'.
INTERNAL-OLIGO-SELF-END is meaningless when
applied to internal oligos used for
hybridization-based detection, since
primer-dimer will not occur. We recommend
that INTERNAL-OLIGO-SELF-END be set at least
as high as INTERNAL-OLIGO-SELF-ANY. (Number
up to 9999.990)
* -oligomaxpolyx integer [5] The maximum allowable length of an
internal oligo mononucleotide repeat, for
example AAAAAA. (Integer 0 or more)
* -oligomaxmishyb float [12.0] Similar to MAX-MISPRIMING except that
this parameter applies to the similarity of
candidate internal oligos to the library
specified in INTERNAL-OLIGO-MISHYB-LIBRARY.
(Number up to 9999.990)
Advanced (Unprompted) qualifiers:
-mispriminglibraryfile infile The name of a file containing a nucleotide
sequence library of sequences to avoid
amplifying (for example repetitive
sequences, or possibly the sequences of
genes in a gene family that should not be
amplified.) The file must be in (a slightly
restricted) FASTA format (W. B. Pearson and
D.J. Lipman, PNAS 85:8 pp 2444-2448 [1988]);
we briefly discuss the organization of this
file below. If this parameter is specified
then EPrimer3 locally aligns each candidate
primer against each library sequence and
rejects those primers for which the local
alignment score times a specified weight
(see below) exceeds MAX-MISPRIMING. (The
maximum value of the weight is arbitrarily
set to 100.0.)
Each sequence entry in the FASTA-format file
must begin with an 'id line' that starts
with '>'. The contents of the id line is
'slightly restricted' in that EPrimer3
parses everything after any optional
asterisk ('*') as a floating point number to
use as the weight mentioned above. If the
id line contains no asterisk then the weight
defaults to 1.0. The alignment scoring
system used is the same as for calculating
complementarity among oligos (e.g.
SELF-ANY). The remainder of an entry
contains the sequence as lines following the
id line up until a line starting with '>'
or the end of the file. Whitespace and
newlines are ignored. Characters 'A', 'T',
'G', 'C', 'a', 't', 'g', 'c' are retained
and any other character is converted to 'N'
(with the consequence that any IUB / IUPAC
codes for ambiguous bases are converted to
'N'). There are no restrictions on line
length.
An empty value for this parameter indicates
that no repeat library should be used.
-explainflag boolean [N] If this flag is true, produce
LEFT-EXPLAIN, RIGHT-EXPLAIN, and
INTERNAL-OLIGO-EXPLAIN output tags, which
are intended to provide information on the
number of oligos and primer pairs that
EPrimer3 examined, and statistics on the
number discarded for various reasons.
-fileflag boolean [N] If the associated value is true, then
EPrimer3 creates two output files for each
input SEQUENCE. File (sequence-id).for lists
all acceptable forward primers for
(sequence-id), and (sequence-id).rev lists
all acceptable reverse primers for
(sequence-id), where (sequence-id) is the
value of the SEQUENCE-ID tag (which must be
supplied). In addition, if the input tag
TASK is 1 or 4, EPrimer3 produces a file
(sequence-id).int, which lists all
acceptable internal oligos.
-firstbaseindex integer [1] This parameter is the index of the first
base in the input sequence. For input and
output using 1-based indexing (such as that
used in GenBank and to which many users are
accustomed) set this parameter to 1. For
input and output using 0-based indexing set
this parameter to 0. (This parameter also
affects the indexes in the contents of the
files produced when the primer file flag is
set.) (Any integer value)
-pickanyway boolean [N] If true pick a primer pair even if
LEFT-INPUT, RIGHT-INPUT, or
INTERNAL-OLIGO-INPUT violates specific
constraints.
-maxmispriming float [12.00] The maximum allowed weighted
similarity with any sequence in
MISPRIMING-LIBRARY. (Number up to 9999.990)
-pairmaxmispriming float [24.00] The maximum allowed sum of weighted
similarities of a primer pair (one
similarity for each primer) with any single
sequence in MISPRIMING-LIBRARY. (Number up
to 9999.990)
-numnsaccepted integer [0] Maximum number of unknown bases (N)
allowable in any primer. (Integer 0 or more)
-selfany float [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 forward
and reverse 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 alignment
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
oligos. (Number from 0.000 to 9999.990)
-selfend float [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 forward and reverse
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'
or
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' to 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. (Number 0.000 or more)
-maxendstability float [9.0] The maximum stability for the five 3'
bases of a forward or reverse primer. Bigger
numbers mean more stable 3' ends. The value
is the maximum delta G for duplex
disruption for the five 3' bases as
calculated using the nearest neighbor
parameters published in Breslauer, Frank,
Bloeker and Marky, Proc. Natl. Acad. Sci.
USA, vol 83, pp 3746-3750. EPrimer3 uses a
completely permissive default value for
backward compatibility (which we may change
in the next release). Rychlik recommends a
maximum value of 9 (Wojciech Rychlik,
'Selection of Primers for Polymerase Chain
Reaction' in BA White, Ed., 'Methods in
Molecular Biology, Vol. 15: PCR Protocols:
Current Methods and Applications', 1993, pp
31-40, Humana Press, Totowa NJ). (Number up
to 1000.000)
Associated qualifiers:
"-sequence" associated qualifiers
-sbegin1 integer Start of each sequence to be used
-send1 integer End of each sequence to be used
-sreverse1 boolean Reverse (if DNA)
-sask1 boolean Ask for begin/end/reverse
-snucleotide1 boolean Sequence is nucleotide
-sprotein1 boolean Sequence is protein
-slower1 boolean Make lower case
-supper1 boolean Make upper case
-sformat1 string Input sequence format
-sdbname1 string Database name
-sid1 string Entryname
-ufo1 string UFO features
-fformat1 string Features format
-fopenfile1 string Features file name
"-outfile" associated qualifiers
-odirectory2 string Output directory
General qualifiers:
-auto boolean Turn off prompts
-stdout boolean Write standard output
-filter boolean Read standard input, write standard output
-options boolean Prompt for standard and additional values
-debug boolean Write debug output to program.dbg
-verbose boolean Report some/full command line options
-help boolean Report command line options. More
information on associated and general
qualifiers can be found with -help -verbose
-warning boolean Report warnings
-error boolean Report errors
-fatal boolean Report fatal errors
-die boolean Report dying program messages
</pre>
</td></tr></table>
<P>
<table border cellspacing=0 cellpadding=3 bgcolor="#ccccff">
<tr bgcolor="#FFFFCC">
<th align="left" colspan=2>Standard (Mandatory) qualifiers</th>
<th align="left">Allowed values</th>
<th align="left">Default</th>
</tr>
<tr>
<td>[-sequence]<br>(Parameter 1)</td>
<td>The sequence from which to choose primers. The sequence must be presented 5' to 3'</td>
<td>Readable sequence(s)</td>
<td><b>Required</b></td>
</tr>
<tr>
<td>[-outfile]<br>(Parameter 2)</td>
<td>Whitehead primer3_core program output file</td>
<td>Output file</td>
<td><i><*></i>.eprimer3</td>
</tr>
<tr bgcolor="#FFFFCC">
<th align="left" colspan=2>Additional (Optional) qualifiers</th>
<th align="left">Allowed values</th>
<th align="left">Default</th>
</tr>
<tr>
<td>-[no]primer</td>
<td>Tell EPrimer3 to pick primer(s)</td>
<td>Toggle value Yes/No</td>
<td>Yes</td>
</tr>
<tr>
<td>-task</td>
<td>Tell EPrimer3 what task to perform. Legal values are 1: 'Pick PCR primers', 2: 'Pick forward primer only', 3: 'Pick reverse primer only', 4: 'No primers needed'.</td>
<td><table><tr><td>1</td> <td><i>(Pick PCR primers)</i></td></tr><tr><td>2</td> <td><i>(Pick forward primer only)</i></td></tr><tr><td>3</td> <td><i>(Pick reverse primer only)</i></td></tr><tr><td>4</td> <td><i>(No primers needed)</i></td></tr></table></td>
<td>1</td>
</tr>
<tr>
<td>-hybridprobe</td>
<td>An 'internal oligo' is intended to be used as a hybridization probe (hyb probe) to detect the PCR product after amplification.</td>
<td>Toggle value Yes/No</td>
<td>No</td>
</tr>
<tr>
<td>-oligomishyblibraryfile</td>
<td>Similar to MISPRIMING-LIBRARY, except that the event we seek to avoid is hybridization of the internal oligo to sequences in this library rather than priming from them.
The file must be in (a slightly restricted) FASTA format (W. B. Pearson and D.J. Lipman, PNAS 85:8 pp 2444-2448 [1988]); we briefly discuss the organization of this file below. If this parameter is specified then EPrimer3 locally aligns each candidate oligo against each library sequence and rejects those primers for which the local alignment score times a specified weight (see below) exceeds INTERNAL-OLIGO-MAX-MISHYB. (The maximum value of the weight is arbitrarily set to 12.0.)
Each sequence entry in the FASTA-format file must begin with an 'id line' that starts with '>'. The contents of the id line is 'slightly restricted' in that EPrimer3 parses everything after any optional asterisk ('*') as a floating point number to use as the weight mentioned above. If the id line contains no asterisk then the weight defaults to 1.0. The alignment scoring system used is the same as for calculating complementarity among oligos (e.g. SELF-ANY). The remainder of an entry contains the sequence as lines following the id line up until a line starting with '>' or the end of the file. Whitespace and newlines are ignored. Characters 'A', 'T', 'G', 'C', 'a', 't', 'g', 'c' are retained and any other character is converted to 'N' (with the consequence that any IUB / IUPAC codes for ambiguous bases are converted to 'N'). There are no restrictions on line length.
An empty value for this parameter indicates that no library should be used.</td>
<td>Input file</td>
<td><b>Required</b></td>
</tr>
<tr>
<td>-numreturn</td>
<td>The maximum number of primer pairs to return. Primer pairs returned are sorted by their 'quality', in other words by the value of the objective function (where a lower number indicates a better primer pair). Caution: setting this parameter to a large value will increase running time.</td>
<td>Integer 0 or more</td>
<td>5</td>
</tr>
<tr>
<td>-includedregion</td>
<td>A sub-region of the given sequence in which to pick primers. For example, often the first dozen or so bases of a sequence are vector, and should be excluded from consideration. The value for this parameter has the form
(start),(end)
where (start) is the index of the first base to consider, and (end) is the last in the primer-picking region.</td>
<td>Sequence range</td>
<td><i>full sequence</i></td>
</tr>
<tr>
<td>-target</td>
<td>If one or more Targets is specified then a legal primer pair must flank at least one of them. A Target might be a simple sequence repeat site (for example a CA repeat) or a single-base-pair polymorphism. The value should be a space-separated list of
(start),(end)
pairs where (start) is the index of the first base of a Target, and (end) is the last
E.g. 50,51 requires primers to surround the 2 bases at positions 50 and 51.</td>
<td>Sequence range</td>
<td><i>full sequence</i></td>
</tr>
<tr>
<td>-excludedregion</td>
<td>Primer oligos may not overlap any region specified in this tag. The associated value must be a space-separated list of
(start),(end)
pairs where (start) is the index of the first base of the excluded region, and and (end) is the last. This tag is useful for tasks such as excluding regions of low sequence quality or for excluding regions containing repetitive elements such as ALUs or LINEs.
E.g. 401,407 68,70 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 68.</td>
<td>Sequence range</td>
<td><i>full sequence</i></td>
</tr>
<tr>
<td>-forwardinput</td>
<td>The sequence of a forward primer to check and around which to design reverse primers and optional internal oligos. Must be a substring of SEQUENCE.</td>
<td>Any string is accepted</td>
<td><i>An empty string is accepted</i></td>
</tr>
<tr>
<td>-reverseinput</td>
<td>The sequence of a reverse primer to check and around which to design forward primers and optional internal oligos. Must be a substring of the reverse strand of SEQUENCE.</td>
<td>Any string is accepted</td>
<td><i>An empty string is accepted</i></td>
</tr>
<tr>
<td>-gcclamp</td>
<td>Require the specified number of consecutive Gs and Cs at the 3' end of both the forward and reverse primer. (This parameter has no effect on the internal oligo if one is requested.)</td>
<td>Integer 0 or more</td>
<td>0</td>
</tr>
<tr>
<td>-osize</td>
<td>Optimum length (in bases) of a primer oligo. EPrimer3 will attempt to pick primers close to this length.</td>
<td>Integer 0 or more</td>
<td>20</td>
</tr>
<tr>
<td>-minsize</td>
<td>Minimum acceptable length of a primer. Must be greater than 0 and less than or equal to MAX-SIZE.</td>
<td>Integer 1 or more</td>
<td>18</td>
</tr>
<tr>
<td>-maxsize</td>
<td>Maximum acceptable length (in bases) of a primer. Currently this parameter cannot be larger than 35. This limit is governed by the maximum oligo size for which EPrimer3's melting-temperature is valid.</td>
<td>Integer up to 35</td>
<td>27</td>
</tr>
<tr>
<td>-otm</td>
<td>Optimum melting temperature(Celsius) for a primer oligo. EPrimer3 will try to pick primers with melting temperatures are close to this temperature. The oligo melting temperature formula in EPrimer3 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 discussion.</td>
<td>Any numeric value</td>
<td>60.0</td>
</tr>
<tr>
<td>-mintm</td>
<td>Minimum acceptable melting temperature(Celsius) for a primer oligo.</td>
<td>Any numeric value</td>
<td>57.0</td>
</tr>
<tr>
<td>-maxtm</td>
<td>Maximum acceptable melting temperature(Celsius) for a primer oligo.</td>
<td>Any numeric value</td>
<td>63.0</td>
</tr>
<tr>
<td>-maxdifftm</td>
<td>Maximum acceptable (unsigned) difference between the melting temperatures of the forward and reverse primers.</td>
<td>Any numeric value</td>
<td>100.0</td>
</tr>
<tr>
<td>-ogcpercent</td>
<td>Primer optimum GC percent.</td>
<td>Any numeric value</td>
<td>50.0</td>
</tr>
<tr>
<td>-mingc</td>
<td>Minimum allowable percentage of Gs and Cs in any primer.</td>
<td>Any numeric value</td>
<td>20.0</td>
</tr>
<tr>
<td>-maxgc</td>
<td>Maximum allowable percentage of Gs and Cs in any primer generated by Primer.</td>
<td>Any numeric value</td>
<td>80.0</td>
</tr>
<tr>
<td>-saltconc</td>
<td>The millimolar concentration of salt (usually KCl) in the PCR. EPrimer3 uses this argument to calculate oligo melting temperatures.</td>
<td>Any numeric value</td>
<td>50.0</td>
</tr>
<tr>
<td>-dnaconc</td>
<td>The nanomolar concentration of annealing oligos in the PCR. EPrimer3 uses this argument to calculate oligo melting temperatures. The default (50nM) works well with the standard protocol used at the Whitehead/MIT Center for Genome Research--0.5 microliters of 20 micromolar concentration for each primer oligo in a 20 microliter reaction with 10 nanograms template, 0.025 units/microliter Taq polymerase in 0.1 mM each dNTP, 1.5mM MgCl2, 50mM KCl, 10mM Tris-HCL (pH 9.3) using 35 cycles with an annealing temperature of 56 degrees Celsius. This parameter corresponds to 'c' in Rychlik, Spencer and Rhoads' equation (ii) (Nucleic Acids Research, vol 18, num 12) where a suitable value (for a lower initial concentration of template) is 'empirically determined'. The value of this parameter is less than the actual concentration of oligos in the reaction because it is the concentration of annealing oligos, which in turn depends on the amount of template (including PCR product) in a given cycle. This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures.
See ADVICE FOR PICKING PRIMERS.</td>
<td>Any numeric value</td>
<td>50.0</td>
</tr>
<tr>
<td>-maxpolyx</td>
<td>The maximum allowable length of a mononucleotide repeat in a primer, for example AAAAAA.</td>
<td>Integer 0 or more</td>
<td>5</td>
</tr>
<tr>
<td>-productosize</td>
<td>The optimum size for the PCR product. 0 indicates that there is no optimum product size.</td>
<td>Integer 0 or more</td>
<td>200</td>
</tr>
<tr>
<td>-productsizerange</td>
<td>The associated values specify the lengths of the product that the user wants the primers to create, and is a space separated list of elements of the form
(x)-(y)
where an (x)-(y) pair is a legal range of lengths for the product. For example, if one wants PCR products to be between 100 to 150 bases (inclusive) then one would set this parameter to 100-150. If one desires PCR products in either the range from 100 to 150 bases or in the range from 200 to 250 bases then one would set this parameter to 100-150 200-250.
EPrimer3 favors ranges to the left side of the parameter string. EPrimer3 will return legal primers pairs in the first range regardless the value of the objective function for these pairs. Only if there are an insufficient number of primers in the first range will EPrimer3 return primers in a subsequent range.</td>
<td>Sequence range</td>
<td>100-300</td>
</tr>
<tr>
<td>-productotm</td>
<td>The optimum melting temperature for the PCR product. 0 indicates that there is no optimum temperature.</td>
<td>Any numeric value</td>
<td>0.0</td>
</tr>
<tr>
<td>-productmintm</td>
<td>The minimum allowed melting temperature of the amplicon. Please see the documentation on the maximum melting temperature of the product for details.</td>
<td>Any numeric value</td>
<td>-1000000.0</td>
</tr>
<tr>
<td>-productmaxtm</td>
<td>The maximum allowed melting temperature of the amplicon. Product Tm is calculated using the formula from Bolton and McCarthy, PNAS 84:1390 (1962) as presented in Sambrook, Fritsch and Maniatis, Molecular Cloning, p 11.46 (1989, CSHL Press).
Tm = 81.5 + 16.6(log10([Na+])) + .41*(%GC) - 600/length
Where [Na+} is the molar sodium concentration, (%GC) is the percent of Gs and Cs in the sequence, and length is the length of the sequence.
A similar formula is used by the prime primer selection program in GCG http://www.gcg.com), which instead uses 675.0/length in the last term (after F. Baldino, Jr, M.-F. Chesselet, and M.E. Lewis, Methods in Enzymology 168:766 (1989) eqn (1) on page 766 without the mismatch and formamide terms). The formulas here and in Baldino et al. assume Na+ rather than K+. According to J.G. Wetmur, Critical Reviews in BioChem. and Mol. Bio. 26:227 (1991) 50 mM K+ should be equivalent in these formulae to .2 M Na+. EPrimer3 uses the same salt concentration value for calculating both the primer melting temperature and the oligo melting temperature. If you are planning to use the PCR product for hybridization later this behavior will not give you the Tm under hybridization conditions.</td>
<td>Any numeric value</td>
<td>1000000.0</td>
</tr>
<tr>
<td>-oligoexcludedregion</td>
<td>Middle oligos may not overlap any region specified by this tag. The associated value must be a space-separated list of
(start),(end)
pairs, where (start) is the index of the first base of an excluded region, and (end) is the last. Often one would make Target regions excluded regions for internal oligos.</td>
<td>Sequence range</td>
<td><i>full sequence</i></td>
</tr>
<tr>
<td>-oligoinput</td>
<td>The sequence of an internal oligo to check and around which to design forward and reverse primers. Must be a substring of SEQUENCE.</td>
<td>Any string is accepted</td>
<td><i>An empty string is accepted</i></td>
</tr>
<tr>
<td>-oligoosize</td>
<td>Optimum length (in bases) of an internal oligo. EPrimer3 will attempt to pick primers close to this length.</td>