You can set the parameters
listed below from the command
line. For more information,
see "Using Program Parameters" in Chapter 3,
Using Programs in the User's
Guide.
-
-
-MINPRImer=18
-
specifies the minimum primer length.
-
-MAXPRImer=22
-
specifies the maximum primer length.
-
-MINPROduct=100
-
specifies the minimum PCR product
length (when choosing PCR products).
-
-MAXPROduct=300
-
specifies the maximum PCR product
length (when choosing PCR products).
-
-LIStsize=25
-
sets the maximum number of
primers or PCR primer pairs
you want to save in
the output file.
By default, 25 primers or
PCR primer pairs are saved
in the output file.
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
-FORwardprimers, -REVerseprimers, 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
-
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
-
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.
-
-FORwardprimers
-
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.
-
-REVerseprimers
-
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.
-
-NOPROducts
-
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
-
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
-
specifies an input file of
primer sequences from which to
select appropriate oligonucleotide
primers that bind to the
template sequence and meet all
primer and product constraints.
-
-REPeats=@mylist.list
-
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
-
specifies the nM concentration of
primer DNA in the reaction.
This value is used
in the
calculation of primer melting temperature.
-
-SALtconcentration=50.0
-
specifies the mM salt concentration
in the reaction. This
value is used in the
calculation of both
primer and PCR product melting
temperatures.
-
-CLAmp=S
-
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
-
sets the minimum acceptable %
G+C for oligonucleotide primers.
-
-GCMAXPRImer=55.0
-
sets the maximum acceptable %
G+C for oligonucleotide primers.
-
-TMMINPRImer=50.0
-
sets the minimum acceptable melting
temperature (in degrees Celsius) for
oligonucleotide
primers.
-
-TMMAXPRImer=65.0
-
sets the maximum acceptable melting
temperature (in degrees Celsius) for
oligonucleotide
primers.
-
-ENDANNEALPrimer=8.0
-
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
-
-
-ENDWGTPrimer=2.0
-
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
-
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.
-
-
-ALLWGTPrimer=1.0
-
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
-
sets the minimum acceptable %
G+C for amplified PCR products.
-
-GCMAXPROduct=55.0
-
sets the maximum acceptable %
G+C for amplified PCR products.
-
-TMMINPROduct=70.0
-
sets the minimum acceptable melting
temperature (in degrees Celsius) for
amplified PCR
products.
-
-TMMAXPROduct=95.0
-
sets the maximum acceptable melting
temperature (in degrees Celsius) for
amplified PCR
products.
-
-TMDIFference=2.0
-
sets the maximum acceptable difference
between the melting temperatures of
the two primers in
a PCR amplification experiment.
-
-ENDANNEALTemplate=16.0
-
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
-
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
-
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
-
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.
-
-RSF=prime.rsf
-
writes an RSF (rich sequence
format) file containing the input
sequences annotated with
features generated from the results
of Prime. This RSF
file is suitable for input
to other
Wisconsin Package programs that support
RSF files. In particular,
you can use SeqLab to
view
this features annotation graphically.
If you don't specify a
file name with this parameter,
then
the program creates one using
prime for the file basename
and .rsf for the extension.
For more
information on RSF files, see
"Using Rich Sequence Format (RSF)
Files" in Chapter 2 of
the
User's Guide. Or, see
"Rich Sequence Format (RSF) Files"
in Appendix C of the
SeqLab Guide.
-
-DENsity=1700
-
sets the number of bases
or amino acids per 100
platen units (PU). This
is usually equivalent to
the number of bases or
amino acids per page.
Output from different GCG graphics
programs
that are run at the
same density can be compared
by lining up the plots
on a light box.
-
-SPAcing=1.6
-
sets the spacing between each
line of the display to
1.6 platen units. If
the plot seems crowded
on your plotter, try setting
the spacing to 3.0.
-
-NOPLOt
-
suppresses the plot of primer
site locations.
-
-MONitor
-
This program normally monitors its
progress on your screen.
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.
-
-SUMmary
-
writes a summary of the
program's work to the screen
when you've used -Default to
suppress
all program interaction. A
summary typically displays at the
end of a program run
interactively.
You can suppress the summary
for a program run interactively
with -NOSUMmary.
You can also use this
parameter to cause a summary
of the program's work to
be written in the
log file of a program
run in batch.
-
-BATch
-
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 Wisconsin Package graphics programs.
These and many others
are
described in detail in Chapter 5, Using Graphics
of the
User's Guide.
