PRIME+
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Table of Contents
FUNCTION
DESCRIPTION
EXAMPLE
OUTPUT
INPUT
FILES
RELATED
PROGRAMS
RESTRICTIONS
ALGORITHM
CONSIDERATIONS
SUGGESTIONS
GRAPHICS
<CTRL>C
COMMAND-LINE
SUMMARY
LOCAL
DATA FILES
PARAMETER
REFERENCE
FUNCTION
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Prime+ selects oligonucleotide Primers for a
template DNA sequence. The primers may be useful for the polymerase chain
reaction (PCR) or for DNA sequencing. You can allow Prime+ to choose primers
from the whole template or limit the choices to a particular set of primers
listed in a file.
The Polymerase Chain Reaction (PCR) process for
amplifying nucleic acids is covered by U.S.
Patent Nos. 4,683,195 and 4,683,202 owned by Hoffmann La Roche. A license for
research may be obtained through the purchase and use of authorized reagents
and thermocyclers from Perkin-Elmer Corp., or by otherwise negotiating a license
with Perkin-Elmer. No license to use PCR is granted by the purchase or use of
the Accelrys GCG (GCG).
DESCRIPTION
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Advantages of Plus “+” Programs:
P
Plus programs are enhanced to be able to read
sequences in a variety of native formats such as GCG RSF, GCG SSF, GCG MSF,
GenBank, EMBL, FastA, SwissProt, PIR, and BSML without conversion.
P
Plus programs remove sequence length restriction
of 350,000bp.
If you do not need these features and wish to have
more interactivity, you might wish to seek out and run the original program
version.
Prime+ analyzes a template DNA sequence and
chooses primer pairs for the polymerase chain reaction (PCR) and primers for
DNA sequencing. For PCR primer pair selection, you can choose a target range of
the template sequence to be amplified. For DNA sequencing primers, you can
specify positions on the template that must be included in the sequencing.
In selecting appropriate primers, Prime+
considers a variety of constraints on the primer and amplified product
sequences. You either can use the program's default constraint values, ignore
all constraints or modify those values to customize the analysis. You can
specify upper and lower limits for primer and product melting temperatures and
for primer and product GC contents. For primers, you can specify a range of
acceptable primer sizes, any required bases at the 3' end of the primer (3' clamp), and a maximum difference in
primer melting temperatures for PCR primer pairs. For PCR products, you can
specify a range of acceptable product sizes.
For efficient priming, you should avoid primers
with extensive self-complementarity in order to minimize primer secondary
structure and primer dimer formation. Additionally, in PCR experiments, primer
pairs with extensive complementarity between the two primers should be avoided
in order to minimize primer dimer formation. Prime+ uses the annealing test
described in the ALGORITHM topic to check individual primers for
self-complementarity and to check the two primers in a PCR primer pair for
complementarity to each other. Using this same annealing test, Prime+
optionally can screen against non-specific primer binding on the template
sequence and on any repeated sequences you specify.
The terms forward primer and reverse
primer are used in the remainder of this document and in the program
output. Forward primers are complementary to sequences on the reverse template
strand and create copies of the forward strand by primer extension. Conversely,
reverse primers are complementary to sequences on the forward template strand
and create copies of the reverse strand by primer extension.
EXAMPLE
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Here is a session using Prime+ to select PCR
primers to amplify a the sequence of a human globin DNA sequence.
prime+
Prime+ selects oligonucleotide primers for a template DNA sequence. The primers may be useful for the polymerase chain reaction (PCR) or for DNA sequencing. You can allow Prime+ to choose primers from the whole template or limit the choices to a particular set of primers listed in a file. The Polymerase Chain Reaction (PCR) process for amplifying nucleic acids is covered by U.S. Patent Nos. 4,683,195 and 4,683,202 owned by Hoffmann La Roche. A license for research may be obtained through the purchase and use of authorized reagents and thermocyclers from Perkin-Elmer Corp., or by otherwise negotiating a license with Perkin-Elmer. No license to use PCR is granted by the purchase or use of Basilisk.
prime+ of what sequence(s) ? ggamma.seq
What should I call the output file (* <sequence_name>.prime+ *) ?
begin1 (* 1 *) ?
End (-1 for entire sequence) (* -1 *) ?
minprimer (* 18 *) ?
maxprimer (* 22 *) ?
minproduct (* 100 *) ?
maxproduct (* 300 *) ?
Analyzing sequence 'ggamma.seq' from 'ggamma.seq'
Running forward primer search...
Search is 1.00% complete
Search is 55.18% complete
Search is 100.00% complete
Running reverse primer search...
Search is 1.00% complete
Search is 76.00% complete
Search is 100.00% complete
Running product search...
Search is 0.00% complete
Search is 100.00% complete
Processing product search results...
Results written to ggamma.prime+
OUTPUT
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INPUT
SUMMARY
-------------
Primer constraints:
primer size: 18 - 22
primer 3' clamp: S
primer sequence ambiguity:
NOT ALLOWED
primer GC content: 40.0 -
55.0%
primer Tm: 50.0 - 65.0 degrees Celsius
primer self-annealing. . .
3' end:
< 8
(weight: 2.0)
total: < 14
(weight: 1.0)
unique primer binding sites:
required
primer-template and
primer-repeat annealing. . .
3' end:
ignored
total: ignored
repeated sequences screened:
none specified
Product constraints:
product length: 100 - 300
product GC content: 40.0 -
55.0%
product Tm: 70.0 - 95.0
degrees Celsius
duplicate primer endpoints:
NOT ALLOWED
difference in primer Tm:
< 2.0 degrees Celsius
primer-primer annealing. . .
3' end:
< 8
(weight: 2.0)
total: < 14
(weight: 1.0)
Maximum overlap between
products: 300 bp
[DNA] = 50.000 nM [salt] = 50.000 mM
PRIME
of: ggamma.seq ck: 7694 from: 1 to: 1699 December
03, 2004 16:46
PRIMER SUMMARY
--------------
forward
reverse
Number of primers considered:
4452
4520
Number of primers rejected for . . .
primer 3' clamp:
906
903
primer sequence ambiguity:
0
0
primer GC content:
1769
1777
primer Tm:
814
819
non-unique
binding sites:
0
2
primer self-annealing:
194
167
primer-template
annealing:
0
0
primer-repeat annealing:
0
0
Number
of primers accepted:
769
852
Number of primers saved:
769
852
PRODUCT SUMMARY
---------------
Number of products considered:
655188
Number of products rejected for. . .
product length:
578989
product GC content:
2960
product Tm:
13
product position:
0
duplicate primer endpoints: 29282
difference
in primer Tm:
16779
primer-primer annealing:
21744
product overlap:
0
Number of products accepted:
5421
Number of products
saved:
25
--------------------------------------------------------------------------------
Product: 1
PRIMERS
-------
5'
3'
forward primer
(20-mer):
837 TGGAGGCAGGACAAGTATGG 856
reverse primer
(19-mer): 1106
CACACACACACACACACAC 1088
forward
reverse
primer %GC:
55.0
52.6
primer Tm (degrees Celsius):
53.2
51.8
PRODUCT
-------
product length: 270
product %GC: 42.6
product Tm: 74.9 degrees
Celsius
difference
in primer Tm: 1.4 degrees
Celsius
annealing score:
27
optimal annealing temperature: 53.0 degrees Celsius
--------------------------------------------------------------------------------
Product: 2
PRIMERS
-------
5'
3'
forward primer
(19-mer):
863 AAAAGATGCAGGCAGAAGG 881
reverse primer
(18-mer): 1105
ACACACACACACACACAC 1088
forward
reverse
primer %GC:
47.4
50.0
primer Tm (degrees Celsius):
50.0
50.1
PRODUCT
-------
product length: 243
product %GC: 41.6
product Tm: 74.2 degrees
Celsius
difference
in primer Tm: 0.1 degrees
Celsius
annealing score:
27
optimal annealing temperature: 52.0 degrees Celsius
--------------------------------------------------------------------------------
Product: 16
PRIMERS
-------
5'
3'
forward primer
(18-mer):
530 TGTTTCAGCACTGTTGCC 547
reverse primer
(22-mer):
753 CCTCATTTCCACCTCTCTTTTC 732
forward
reverse
primer %GC:
50.0
45.5
primer Tm (degrees Celsius):
50.5
50.9
PRODUCT
-------
product length: 224
product %GC: 46.9
product Tm: 76.1 degrees
Celsius
difference
in primer Tm: 0.4 degrees
Celsius
annealing
score: 32
optimal annealing temperature: 53.5 degrees Celsius
--------------------------------------------------------------------------------
Product: 17
PRIMERS
-------
5'
3'
forward primer
(20-mer):
535 CAGCACTGTTGCCTTTAGTC
554
reverse primer
(22-mer):
760 TCATTTTCCTCATTTCCACCTC 739
forward
reverse
primer %GC:
50.0
40.9
primer Tm (degrees Celsius):
51.4
50.4
PRODUCT
-------
product length: 226
product %GC: 46.5
product Tm: 76.0 degrees
Celsius
difference
in primer Tm: 0.9 degrees
Celsius
annealing score:
32
optimal annealing temperature: 53.4 degrees Celsius
--------------------------------------------------------------------------------
Product: 18
PRIMERS
-------
5'
3'
forward primer
(20-mer):
535 CAGCACTGTTGCCTTTAGTC
554
reverse primer
(22-mer):
758 ATTTTCCTCATTTCCACCTCTC 737
forward
reverse
primer %GC:
50.0
40.9
primer Tm (degrees Celsius):
51.4
50.1
PRIMERS
-------
5'
3'
forward primer
(20-mer): 837
TGGAGGCAGGACAAGTATGG 856
reverse primer
(20-mer): 1133
TCACACACACACAAACACAC 1114
forward
reverse
primer %GC:
55.0
45.0
primer Tm (degrees Celsius):
53.2
51.4
PRODUCT
-------
product length: 297
product %GC: 43.8
product Tm: 75.6 degrees
Celsius
difference in primer
Tm: 1.8 degrees Celsius
annealing score:
32
optimal annealing temperature: 53.4 degrees Celsius
--------------------------------------------------------------------------------
Product: 21
PRIMERS
-------
5'
3'
forward primer
(20-mer):
837 TGGAGGCAGGACAAGTATGG 856
reverse primer
(19-mer): 1104
CACACACACACACACACAG 1086
forward
reverse
primer %GC:
55.0
52.6
primer Tm (degrees Celsius):
53.2
51.5
PRODUCT
-------
product length: 268
product %GC: 42.5
product Tm: 74.8 degrees
Celsius
difference
in primer Tm: 1.7 degrees
Celsius
annealing score:
32
optimal annealing temperature: 52.9 degrees Celsius
--------------------------------------------------------------------------------
Product: 22
PRIMERS
-------
5'
3'
forward primer
(20-mer):
837 TGGAGGCAGGACAAGTATGG 856
reverse primer
(20-mer): 1103
ACACACACACACACACAGAG 1084
forward
reverse
primer
%GC:
55.0
50.0
primer Tm (degrees Celsius):
53.2
52.6
--------------------------------------------------------------------------------
INPUT
FILES
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Prime+
accepts any nucleotide sequence as input and selects appropriate
oligonucleotide primers that are complementary to sites on the input template
sequence. If Prime+ rejects your nucleotide sequence, turn to Appendix VI to see how to change or set the type of
a sequence.
You
optionally can specify one or two input files of primer sequences from which to
select appropriate oligonucleotide primers that are complementary to sites on
the template sequence with -PRImers
or -PRIMERSF
and/or
-PRIMERSR. The file of primer sequences for Prime+ is modeled
on the enzyme data files for the mapping programs described in Appendix VII. The primer names should not have
more than 31 characters. The offset field is ignored by Prime+, but the field
must have a number in it to make the input primer files compatible with the
files that are read by mapping programs. The input primer sequence may contain
only valid GCG sequence symbols (see Appendix III
of this manual) and the single quotation mark ( ' ) and underscore ( _ )
characters. Single quotation marks and underscores in the sequence patterns are
ignored. Prime+ ignores input primers containing any other characters in the
sequence. The overhang field has no significance to Prime+ and can be omitted.
For other GCG mapping programs, if the overhang field is absent or is a
non-numeric character, then the bottom strand is not searched.
The
exact spacing between each field does not matter, only the order of the fields
in the line. Blank lines and lines that start with an exclamation point
("!") are ignored. Here is part of an example file of input primers:
An example file of input primers for the PRIME program.
Name Offset Sequence Documentation ..
x13598 1 ACCCTTCAGCAGTTCCACAC !
x24332 1 AAGCACCCTTCAGCAGTTCC !
u35982 1 AAGAGAGGTGGAAATGAGG !
RELATED PROGRAMS
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The
GCG mapping programs Map,Map+, MapPlot,
and MapSort can be used to mark finds in the context
of a DNA restriction map. FindPatterns+
identifies sequences that contain short patterns like GAATTC or YRYRYRYR. You
can define the patterns ambiguously and allow mismatches. You can provide the
patterns in a file or simply type them in from the terminal.
Prime selects oligonucleotide Primers for a template DNA
sequence. The primers may be useful for the polymerase chain reaction (PCR) or
for DNA sequencing. You can allow Prime+ to choose primers from the whole
template or limit the choices to a particular set of primers listed in a file.
RESTRICTIONS
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You
cannot search for primers longer than 100 bases. You cannot specify a maximum
product length greater than 10,000 bases. Prime+ will not read more than 5,000
primers from an input file of primer sequences.
ALGORITHM
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Thermodynamic
Calculations
Prime+
determines primer melting temperatures by a calculation using the
nearest-neighbor model of Borer, and thermodynamic parameters for DNA
nearest-neighbor interactions and the salt dependence of oligonucleotides
determined by SantaLucia (Proc. Natl. Acad. Sci. USA. 95;
1460-1465 (1986)):
T(m)(primer) = delta H / ((delta S' + R x ln(c/4)) - 273.15
where
delta H is the enthalpy of helix formation, delta S' is the salt-adjusted entropy of helix formation
(including helix initiation), R is the molar gas constant (1.987 cal/degree
Celsius/mol), and c is the primer concentration.
In
the above equation, the salt-adjusted delta S'
is determined from the delta S at 1M salt according to the equation:
delta S' = delta S + (0.368 x (primer_length - 1) x ln[K(+)])
where
[K(+)] is the potassium ion concentration.
Prime+
determines PCR product melting temperatures using the formula of Baldino, et
al. (in Methods Enzymol. 168; 761-777 (1989)) as modified
slightly by Rychlik, et al. (Nucleic Acids Res. 18; 6409-6412
(1990)).
T(m)(product) = 0.41 x (% G+C) + 16.6 x log[K(+)] - 675 / len + 81.5
where
len is the length of the product.
If
you are selecting PCR primer pairs, the output includes a proposed annealing
temperature for each listed primer pair. The annealing temperature is
calculated using the formula of Rychlik, et al. (Nucleic Acids Res. 18;
6409-6412 (1990)).
T(a) = 0.3 x T(m)(primer) + 0.7 x T(m)(product) - 14.9
Annealing
Tests
Prime+
uses an annealing test described by Hillier and Green (PCR Methods and
Applications. 1; 124-128 (1991)), with slight modification, to
check individual primers for self-complementarity and to check the two primers
in a PCR primer pair for complementarity to each other. For tests of
self-complementarity, a primer sequence in the 5' to 3'
orientation is compared with the same sequence in the 3' to 5'
orientation. For tests of complementarity between two different primers, one of
the primer sequences in the 5'
to 3' orientation is compared
to the other sequence in the 3'
to 5' orientation. The
sequences are compared in every register of comparison, using a scoring matrix
containing values of complementarity for every pair of nucleotide symbols. (See
the LOCAL DATA FILES topic for more information on the scoring matrix.) For
each register of comparison, the score of each base pair comparison is
determined. The scores of contiguous base pairs with positive comparison values
are summed. The maximum score of all such contiguous segments, taken over all
registers of comparison between the sequences, determines the total
primer-primer annealing score. Complementarity at the 3' ends of the primer sequences has a
particularly large influence on primer-dimer formation. Therefore, the maximum
score of all contiguous segments that include the 3' position of either primer sequence, taken over all
registers of comparison, is separately determined as the 3' primer-primer annealing score.
The
same annealing test is used to determine complementarity between the primer and
any non-specific binding sites on the template sequences. In this case, the
primer in the 5' to 3' orientation is compared over all
registers of comparison with both strands of the template sequence in the 3' to 5'
orientation to determine a total primer-template annealing score. Since
complementarity at the 3' end
of the primer sequence has a particularly large effect on non-specific primer
binding, the 3'
primer-template annealing score is also determined. If you screen against
non-specific primer binding on any specified repeated sequences, then total primer-repeat
and 3' primer-repeat annealing
scores, taken over all registers of comparison in all repeated sequences, are
also determined.
Total
and 3' annealing scores are
saved in tests of primer self-complementarity (to check for secondary structure
and primer dimer formation) and in tests of complementarity between the two
primers in PCR primer pairs (to check for primer dimer formation). Total and 3' annealing scores are also saved when
you screen against non-specific primer binding on the template sequence and
when you screen against non-specific primer binding on any specified repeated
sequences. Primers are rejected that exceed the maximum score you specify for
any of these tests. For those primers that are accepted, the program uses the
sum of all annealing scores to determine the order of primers or PCR primer
pairs in the output list. You can specify weights for each of these scores to
adjust their relative contributions in determining the output order. By
default, 3' annealing scores
have twice the weight of total annealing scores in determining the output
order.
CONSIDERATIONS
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The
template sequence may contain ambiguous bases, but Prime+ will not select
primers complementary to any ambiguous sites on the template sequence. If you
specify an input file of primer sequences from which to select appropriate
oligonucleotide primers, Prime+ will not select any primers in the file that
contain ambiguous bases.
By
default, primer selects appropriate PCR primer pairs. To search for DNA
sequencing primers, you need to use either -forward or -reverse.
When
several acceptable PCR primer pairs have the same 3' ends for both primers, Prime+ outputs only the PCR
primer pair with the shortest primer sequences. By not allowing duplicate
primer endpoints, Prime+ increases the diversity among the PCR primer pairs in
the output list.
Prime+
only determines melting temperatures for DNA primers. We do not know of any
appropriate nearest-neighbor thermodynamic parameters for RNA-DNA hybrids, so
we haven't attempted to calculate melting temperatures for RNA primers. While
thermodynamic parameters for RNA duplexes involving mismatches have been
described, we do not know of any similar results for DNA duplexes. Therefore,
we have not attempted to calculate melting temperatures or other thermodynamic
properties for DNA duplexes involving mismatches.
Prime+
does not currently consider formamide concentration in determining primer
melting temperatures.
SUGGESTIONS
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If
Prime+ fails to select any appropriate primers or PCR primer pairs, review the
program summary displayed both on the terminal screen and in the output file.
This summary lists the number of primers and PCR primer pairs rejected because
they failed to meet each program constraint. With this information, you can
determine which constraints to relax in subsequent runs of the Prime+ program.
To
avoid reporting trivially different PCR primer pairs in the output list, use -maxoverlap
to select sets of primer pairs whose PCR products have limited overlap with
each other.
GRAPHICS
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Accelrys GCG (GCG) must be configured for graphics before you run any
program with graphics output! If the %
setplot
command is available in your installation, this is the easiest way to establish
your graphics configuration, but you can also use commands like % postscript that correspond to the
graphics languages GCG supports. See Section 5, Using Graphics in the User's
Guide for more information about configuring your process for graphics.
<CTRL>C
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If
you need to stop this program, use <Ctrl>C to reset your terminal and
session as gracefully as possible. Searches and comparisons write out the
results from the part of the search that is complete when you use
<Ctrl>C. The graphics device should stop plotting the current page and
start plotting the next page. If the current page is the last page, plotters
should put the pen away and graphic terminals should return to interactive
mode.
COMMAND-LINE SUMMARY
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All
parameters for this program may be added to the command line. Use -check to
view the summary below and to specify parameters before the program executes. In
the syntax summary below, square brackets ([ and ]) enclose parameter values
that are optional. For each program parameter, square brackets enclose the type
of parameter value specified, the default parameter value, and shortened forms
of the parameter name, aliases.
Programs with a plus in the name use either the full parameter name or a
specified alias. If “Type” is “Boolean”, then the
presence of the parameter on the command line indicates a true condition. A
false condition needs to be stated as, parameter=false.
Prime+
selects oligonucleotide primers for a template DNA sequence. The primers may be
useful for the polymerase chain reaction (PCR) or for DNA sequencing. You can
allow Prime to choose primers from the whole template or limit the choices to a
particular set of primers listed in a file. The Polymerase Chain Reaction (PCR)
process for amplifying nucleic acids is covered by U.S. Patent Nos. 4,683,195
and 4,683,202 owned by Hoffmann La Roche. A license for research may be
obtained through the purchase and use of authorized reagents and thermocyclers
from Perkin-Elmer Corp., or by otherwise negotiating a license with
Perkin-Elmer. No license to use PCR is granted by the purchase or use of GCG.
Minimal
Syntax: % prime+ [-infile=]value [-outfile=]value -Default
Minimal
Parameters (case-insensitive):
-infile
[Type: InFile / Default: EMPTY / Aliases: infile1 in]
The name of the input file.
Prompted
Parameters (case-insensitive):
-outfile
[Type: OutFile / Default: '<sequence_name>.prime+' /
Aliases:out]Names the output file.
-begin
[Type: Integer / Default: '1' / Aliases: beg beg1 begin1]
Sets the start of the range of interest.
-end
[Type: Integer / Default: '-1' / Aliases: end1]
Sets the end of the range of interest (-1 for all).
-minprimer [Type: Integer /
Default: '18' / Aliases: minpri]
Sets the minimum primer length.
-maxprimer [Type: Integer /
Default: '22' / Aliases: maxpri]
Sets the maximum primer length.
-minproduct [Type: Integer /
Default: '100' / Aliases: minpro]
Sets the minimum product length.
-maxproduct [Type: Integer /
Default: '300' / Aliases: maxpro]
Sets the maximum product length.
Optional
Parameters (case-insensitive):
-check
[Type: Boolean / Default: 'false' / Aliases: che help]
Prints out this usage message.
-default
[Type: Boolean / Default: 'false' / Aliases: d def]
Specifies that sensible default values be used for all parameters where
possible.
-documentation [Type: Boolean / Default: 'true' /
Aliases: doc]
Prints banner at program startup.
-quiet
[Type: Boolean / Default: 'false' / Aliases: qui]
Tells application to print only a minimal amount of information.
-matrix
[Type: String / Default: 'share_matrix:prime.cmp' / Aliases:data1
dat1]Assigns scoring matrix for annealing tests.
-enthalpy [Type:
String / Default: 'share_energy:dnastack.dh' / Aliases: data2 dat2]Assigns
enthalpies for DNA melting temperature determination.
-entropy
[Type: String / Default: 'share_energy:dnastack.ds' / Aliases: data3
dat3]Assigns entropies for DNA melting temperature determination.
-listsize [Type:
Integer / Default: '25' / Aliases: lis] Sets the maximum number of output
primers or PCR products
shown.
The default value is changed to 100 if option –RELAx is set. -1 means
all.
-begin2
[Type: Integer / Default: '-1' / Aliases: beg2] Sets the start of the
target range for PCR amplification. If this is set to -1 then it is considered
not set.
-end2
[Type: Integer / Default: '-1'] Sets the end of the range for PCR
amplification (-1 for all).
-include
[Type: Double / Default: '100.0' / Aliases: inc] Sets the minimum % of
specified PCR target range to be included in PCR products.
-maxoverlap [Type: Integer /
Default: '300' / Aliases: maxo]
Specifies maximum overlap among different PCR products. -1 means any amount is allowed.
-forward
[Type: Boolean / Default: 'false' / Aliases: for]
Selects forward primers only.
-reverse
[Type: Boolean / Default: 'false' / Aliases: rev]
Selects reverse primers only.
-products [Type:
Boolean / Default: 'true' / Aliases: pro]
Enables/suppresses selection of PCR products.
-unique
[Type: Boolean / Default: 'true' / Aliases: uni]
If set, primers must bind uniquely on template.
-primers
[Type: String / Default: EMPTY / Aliases: pri]
Assigns input file of forward and reverse primers to consider.
-primersf [Type:
String / Default: EMPTY] Assigns input file of forward primers to consider.
This option can be used in combination with option -primersr, but not in
combination with option –primers.
-primersr [Type:
String / Default: EMPTY] Assigns input file of reverse primers to consider.
This option can be used in combination with option -primersf, but not in
combination with option –primers.
-repeats
[Type: String / Default: EMPTY / Aliases: rep]
Assigns
repeated sequences to check for false priming.
-dnaconcentration
[Type: Double / Default: '50.0' / Aliases: dna] Sets the primer DNA
concentration (nM)
-saltconcentration
[Type: Double / Default: '50.0' / Aliases: sal salt]Sets the salt concentration
(mM)
-clamp
[Type: String / Default: 'S' / Aliases: cla]
Specifies primer 3' clamp (using IUB ambiguity codes).
-gcminprimer [Type: Double / Default:
'40.0' / Aliases: gcminpri]
Sets the minimum primer %G+C.
-gcmaxprimer [Type: Double / Default:
'55.0' / Aliases: gcmaxpri]
Sets the maximum primer %G+C.
-tmminprimer [Type: Double / Default:
'50.0' / Aliases: tmminpri]
Sets the minimum primer melting temperature (Celsius).
-tmmaxprimer [Type: Double / Default:
'65.0' / Aliases: tmmaxpri]
Sets the maximum primer melting temperature (Celsius).
-endannealprimer
[Type: Integer / Default: '8' / Aliases: endannealp]
Sets the maximum primer-primer 3' annealing score.
-allannealprimer
[Type: Integer / Default: '14' / Aliases: allannealp]
Sets the maximum primer-primer annealing score.
-endwgtprimer [Type: Double / Default: '2.0' /
Aliases: endwgtp]
Sets
the relative weight of primer-primer 3' annealing score.
-allwgtprimer [Type: Double / Default: '1.0' /
Aliases: allwgtp]
Sets
the relative weight of primer-primer annealing score.
-gcminproduct [Type: Double / Default: '40.0' /
Aliases: gcminpro]
Sets
the minimum product %G+C.
-gcmaxproduct [Type: Double / Default: '55.0' /
Aliases: gcmaxpro]
Sets the maximum product %G+C.
-tmminproduct [Type: Double / Default: '70.0' /
Aliases: tmminpro]
Sets the minimum product melting temperature (Celsius).
-tmmaxproduct [Type: Double / Default: '95.0' /
Aliases: tmmaxpro]
Sets the maximum product melting temperature (Celsius).
-tmdifference [Type: Double / Default: '2.0' /
Aliases: tmdif]
Sets
the maximum difference between melting temperature of two primers in PCR.
-endannealtemplate
[Type: Integer / Default: '16' / Aliases: endannealt]
Sets the maximum primer-template 3' annealing score.
-allannealtemplate
[Type: Integer / Default: '28' / Aliases: allannealt]
Sets the maximum primer-template annealing score.
-endwgttemplate
[Type: Double / Default: '0.5' / Aliases: endwgtt]
Sets the relative weight of primer-template 3' annealing score.
-allwgttemplate
[Type: Double / Default: '0.25' / Aliases: allwgtt]
Sets the relative weight of primer-template annealing score.
-relax
[Type: Boolean / Default: 'false' / Aliases: rela]
Relaxes
most of the constraints set by default. The following options are relaxed:
minprimer, maxprimer, minproduct, maxproduct, clamp, gcminprimer, gcmaxprimer,
tmminprimer, tmmaxprimer, gcminproduct, gcmaxproduct, tmminproduct,
tmmaxproduct.
-sortbyta [Type:
Boolean / Default: 'false' / Aliases: sor]
Sorts
the final output list of products by their annealing temperature (increasing).
-foundprimers [Type: String / Default: EMPTY /
Aliases: fou]
Creates a pattern data file of found primers.
-batch
[Type: Boolean / Default: 'false']
Allows to submit a job to a batch queue.
-seqout
[Type: OutFile / Default: EMPTY / Aliases: rsf]
Saves primers as features in a sequence file.
-monitor
[Type: Boolean / Default: 'true' / Aliases: mon]
Enables percent-complete messages for algorithm progress monitoring.
LOCAL DATA FILES
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The
files described below supply auxiliary data to this program. The program
automatically reads them from a public data directory unless you either 1) have
a data file with exactly the same name in your current working directory; or 2)
name a file on the command line with an expression like -data1=myfile.dat.
For more information see Section 4, Using Data Files in the User's Guide.
Prime+
reads a scoring matrix from your local directory or the public database to use
in the annealing tests that test for primer secondary structure, primer dimer
formation, and false priming sites on the template and repeated sequence. The
file /$GCGROOT/Share/Energy/prime.cmp assigns G-C, A-T, and G-T
base pairs values of 3, 2, and 1, respectively, with all other pairs valued at
0.
Prime+
reads the files dnastack.ds and dnastack.dh (Local data Dirs: /$GCGROOT/Share/Energy) for the DNA stacking
entropies and enthalpies, respectively, used to calculate oligonucleotide
primer melting temperatures.
PARAMETER REFERENCE
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You
can set the parameters listed below from the command line. Shortened forms of
the parameter name, aliases, are shown, separated by commas. All the criteria
that are set by default can be individually relaxed by prefixing the parameter
name with NO.
-infile, -infile1, -in
The name
of the input file.
-outfile, -out
Names the output file.
-begin, -beg,
-beg1, -begin1
Sets the
start of the range of interest.
-end, -end1
Sets the end of the range of interest (-1 for
all).
-check, -che, -help
Prints
out this usage message.
-default, -def
Specifies that sensible
default values be used for all parameters where possible.
-documentation, -doc
Prints
banner at program startup.
-quiet, -qui
This parameter is not
supported.
-matrix
Assigns
scoring matrix for annealing tests.
-seqout
Saves
primers as features in a sequence file.
-enthalpy
Assigns
enthalpies for DNA melting temperature determination.
-entropy
Assigns entropies for DNA melting temperature determination.
-minprimer=18, -minpri
Specifies the minimum primer length. Use -nominprimer to allow primer
length to be as short as 2 bases.
-maxprimer=22, -maxpri
Specifies
the maximum primer length. Use -nomaxprimer to allow primer length to be as long
as 100 bases.
-minproduct=100, -minpro
Specifies
the minimum PCR product length (when choosing PCR products). Use -nominproduct
to have no lower length limit set.
-maxproduct=300, -maxpro
Specifies
the maximum PCR product length (when choosing PCR products). Use -nomaxproduct
to allow the product length to be as long as 10000 bases.
-listsize=25, -lis
Sets
the maximum number of primers or PCR primer pairs you want to save in the
output file, up to a limit of 2500. The default value is 25 unless -relax is
on the command line, when the default value becomes 100. If you choose primers
from both strands of the template sequence but suppress the selection of PCR
products with
-noproducts, then the maximum number of output primers is saved
for each strand of the template sequence. In this case, forward primers are
listed before the reverse primers in the output file.
-begin2=500 -end2=750,
Sets
the target range of the template sequence to be amplified using PCR. Unless you
specify that only a portion of the target range must be amplified with -include,
the program will choose primer pairs that amplify the entire target range of
the template sequence.
If
you are not choosing primers for PCR amplification experiments (by specifying -forward, -reverse, or -noproducts),
then you can specify parts of the template sequence that must be included in
primer extension experiments (like DNA sequencing) with the appropriate use of -begin2 and -end2. If
you specify a position with -BEGin2, then all
forward primer extensions must include that position. If you specify a position
with -end2,
then all reverse primer extensions must include that position.
-include=60.0, -inc
Tells
the program to choose primer pairs that amplify at least 60% of the target
range specified with
-begin2 and -end2. By default, primer pairs are chosen that
amplify the entire specified target range.
-maxoverlap=300, -maxo
Sets
the maximum length of overlap shared among different PCR products. By default,
the maximum overlap is the maximum product length. Reduce this value to avoid
seeing trivially different primer pairs in your output.
-forward, -for
Tells
the program to select forward primers only. Forward primers bind to sequences
on the reverse template strand and create copies of the forward strand by
primer extension. NOTE: This parameter used to be called -forwardprimers.
In this version this previous parameter name is still supported, but that will
go away in the next version.
-reverse, -rev
Tells
the program to select reverse primers only. Reverse primers bind to sequences
on the forward template strand and create copies of the reverse strand by
primer extension. NOTE: This parameter used to be called -reverseprimers.
In this version this previous parameter name is still supported, but that will
go away in the next version.
-noproducts, -nopro
Suppresses
the selection of primer pairs for PCR amplification experiments. Both forward
and reverse primers are selected, but they are not tested in pair combinations
to determine if they meet the PCR product constraints. The best forward and
reverse primers selected are reported in the output file.
-nounique, -nouni
Permits
selection of primers that are complementary, without mismatch, to more than one
site on the template sequence. By default, primers are selected that have
single primer binding sites on the template sequence. (For more stringent
control over primer binding on the template sequence, use -allannealtemplate
and
-endannealtemplate.)
-primers=myFile.dat, -pri
Specifies
an input file of forward and reverse primer sequences from which to select
appropriate oligonucleotide primers that bind to the template sequence and meet
all primer and product constraints. If this parameter is used, parameters -primersf
and -primersr
cannot be used.
-primersf=myFileF.dat,
-primersf
Specifies
an input file of forward primer sequences from which to select appropriate
oligonucleotide primers that bind to the template sequence and meet all primer
and product constraints. Reverse primers can also be given to the program
through an input file (specified with the -PRIMERSR parameter) or they can be
searched for in the template sequence. This parameter can not be used in
combination with
-primers.
-primersr=myFileR.dat,
-primersr
Specifies
an input file of reverse primer sequences from which to select appropriate
oligonucleotide primers that bind to the template sequence and meet all primer
and product constraints. Forward primers can also be given to the program
through an input file (specified with the -primersf parameter) or they can be
searched for in the template sequence. This parameteroption can not be used in
combination with
-primers.
-repeats=@mylist.list, -rep
Specifies
an input list of repeated sequences to check for false priming sites with
putative primer sequences. You can specify any valid GCG sequence
specification. Primers are rejected that are complementary, without mismatch,
to any repeated sequence for a length equal to the minimum acceptable primer
length. (For more stringent control over primer binding on repeated sequences,
use
-allannealtemplate and -endannealtemplate.)
-dnaconcentration=50.0, -dna
Specifies
the nM concentration of primer DNA in the reaction. This value is used in the
calculation of primer melting temperature. The default value is 50.0 and the
value may range from 0.1 to 50.0.
-salconcentration=50.0, -sal
Specifies
the mM salt concentration in the reaction. This value is used in the
calculation of both primer and PCR product melting temperatures. The default
value is 50.0 and the value may range from 0.1 to 50.0.
-clamp=S, -cla
Specifies
the required 3'-terminal bases
(3' clamp) for the primer. Any
number of 3'-terminal bases
can be specified, and all IUB nucleotide ambiguity symbols (described in Appendix III of the Program
Manual) may be used. For example, -clamp=WSS requires all selected
primers to end in the three-base sequence that has an A or T, followed by a G
or C, then terminated by another G or C. By default, the program requires a G
or C as the 3'-terminal base
of all selected primers. Use -noclamp to permit any 3'-terminal bases in selected primers.
-gcminprimer=40.0, -gcminpri
Sets
the minimum acceptable % G+C for oligonucleotide primers. Use -nogcminprimer
to allow % G+C to be as low as 0.0%.
-gcmaxprimer=55.0, -gcmaxpri
Sets
the maximum acceptable % G+C for oligonucleotide primers. Use -nogcmaxprimer
to allow % G+C to be as high as 100.0%.
-tmminprimer=50.0, -tmminpri
Sets
the minimum acceptable melting temperature (in degrees Celsius) for
oligonucleotide primers. Use -notmminprimer to have no lower limit on the
melting temperature set.
-tmmaxprimer=65.0, -tmmaxpri
Sets
the maximum acceptable melting temperature (in degrees Celsius) for
oligonucleotide primers. Use -notmmaxprimer to have no upper limit on the
melting temperature set.
-endannealprimer=8.0,
-endannealp
Sets
the maximum acceptable 3'-terminal
annealing score between two primers in tests of primer-primer complementarity.
This score is used in tests of primer secondary structure and primer dimer
formation. The default maximum acceptable 3'-terminal
annealing score is 8.0. Set this to a higher value if you want to relax this
constraint. Use
-noendannealprimer to have no upper limit on the primer-primer
3' annealing score set.
-endwgtprimer=2.0, -endwgtp
Sets
the relative contribution of the 3'-terminal
annealing score between two primers in determining the output order of primers
and PCR primer pairs.
-allannealprimer=14.0,
-allannealp
Sets
the maximum acceptable annealing score over the entire lengths of two primers
in tests of primer-primer complementarity. This score is used in tests of
primer secondary structure and primer dimer formation. The default maximum
acceptable annealing score over the entire lengths of two primers is 14.0. Set
this to a higher value if you want to relax this constraint. Use -noallannealprimer
to have no upper limit on the primer-primer annealing score set.
-allwgtprimer=1.0, -allwgtp
Sets
the relative contribution of the annealing score over the entire lengths of two
primers in determining the output order of primers and PCR primer pairs.
-gcminproduct=40.0,
-gcminpro
Sets
the minimum acceptable % G+C for amplified PCR products. Use -nogcminproduct
to allow % G+C to be as low as 0.0%.
-gcmaxproduct=55.0,
-gcmaxpro
Sets
the maximum acceptable % G+C for amplified PCR products. Use -nogcmaxproduct
to allow % G+C to be as high as 100.0%.
-tmminproduct=70.0,
-tmminpro
Sets
the minimum acceptable melting temperature (in degrees Celsius) for amplified
PCR products. Use
-notmminproduct to have no lower limit on the melting
temperature set.
-tmmaxproduct=95.0,
-tmmaxpro
Sets
the maximum acceptable melting temperature (in degrees Celsius) for amplified
PCR products. Use
-notmmaxproduct to have no upper limit on the melting
temperature set.
-tmdifference=2.0, -tmdif
Sets
the maximum acceptable difference between the melting temperatures of the two
primers in a PCR amplification experiment. Use -notmdifference to allow any
difference between the melting temperatures.
-endannealtemplate=16.0,
-endannealt
Sets
the maximum acceptable annealing score between the 3' end of the primer and possible false
priming sites on the template sequence. Any repeated sequences you specify with
-repeats
also are checked for false priming sites. By default, Prime+ does not determine
the annealing score between the 3'
end of the primer and possible false priming sites on the template and repeated
sequences. If you specify -endannealtemplate without any value, then a
value of 16.0 is used. Set this to a higher value if you want to relax this
constraint.
-endwgttemplate=0.5,
-endwgtt
Sets
the relative contribution of the annealing score between the 3' end of the primer and possible false
priming sites on the template and repeated sequences in determining the output
order of PCR primer pairs.
-allannealtemplate=28.0,
-allannealt
Sets
the maximum acceptable annealing score between the entire primer and possible
false priming sites on the template sequence. Any repeated sequences you
specify with
-repeats also
are checked for false priming sites. By default, Prime+ does not determine the
annealing score between the entire primer and possible false priming sites on
the template and repeated sequences. If you specify -allannealtemplate
without any value, then a value of 28.0 is used. Set this to a higher value if
you want to relax this constraint.
-allwgttemplate=0.25,
-allwgtt
Sets
the relative contribution of the annealing score between the entire primer
sequence and possible false priming sites on the template and repeated
sequences in determining the output order of PCR primer pairs.
-relax, -rela
As
far as possible, removes limits for most of the constraints that are set by
default: -minprimer, -maxprimer, -minproduct, -maxproduct, -clamp,
-gcminprimer,
-gcmaxprimer, -tmminprimer, -tmmaxprimer, -gcminproduct, -gcmaxproduct,
-tmminproduct, -tmmaxproduct.
Note:
Setting this option can lead to output containing nonsense results. So check
your output carefully.
-sortbyta, -sor
Sorts
the output list of products by their annealing temperature in increasing order,
instead by their annealing scores. The list of products still contains the
products with the lowest annealing scores found.
-foundprimers=primer.dat, -fou
Creates
a data file that lists the primers that are found. Each individual primer or
each primer associated with one or more PCR products is listed once, along with
its begin and end ranges with respect to the template sequence. For example
forward1 1 TGGAGGCAGGACAAGTATGG ! 837 -> 856
Means
that the primer forward1 has the base sequence TGGAGGCAGGACAAGTATGG, which
corresponds to bases 837 through 856 of the template sequence.
This
file's format is the same as that used for the primer files specified with
parameters -primers, -primersf,
and -primersr.
These files can be used as input to a number of GCG programs: Prime,
PrimePair, MeltTemp, Map+, MapSort,
MapPlot, PeptideMap, PeptideSort, and FindPatterns+.
See the INPUT FILES topic for more information on the format of this data file.
-monitor, -mon
Program
monitors its progress on your screen by displaying a screen trace of progress.
However, when you use -default to suppress all program interaction, you
also suppress the monitor. You can turn it back on with this parameter. If you
are running the program in batch, the monitor will appear in the log file.
-seqout, -rsf
Saves
primers as features in a sequence file.
-batch, -bat
Submits
the program to the batch queue for processing after prompting you for all
required user inputs. Any information that would normally appear on the screen
while the program is running is written into a log file. Whether that log file
is deleted, printed, or saved to your current directory depends on how your
system manager has set up the command that submits this program to the batch
queue. All output files are written to your current directory, unless you
direct the output to another directory when you specify the output file.
When
Prime+ is run in batch using -batch, instructions for plotting the primer
sites are written to a figure file named Prime+.figure unless the plot has been
directed to a specific file or graphics device from the command line, or has
been suppressed with
-noplot.
The
parameters below apply to all GCG graphics programs. These and many others
are described in detail in Section 5, Using Graphics of the User's Guide.
Printed:
May 27, 2005 14:06
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Support: support-us@accelrys.com, support-japan@accelrys.com,
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Copyright (c) 1982-2005 Accelrys Inc. All rights reserved.
Licenses and Trademarks: Discovery Studio ®,
SeqLab ®, SeqWeb ®, SeqMerge ®, GCG ® and, the GCG logo are
registered trademarks of Accelrys Inc.
All other product names mentioned in this
documentation may be trademarks, and if so, are trademarks or registered
trademarks of their respective holders and are used in this documentation for
identification purposes only.
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