THE SOFTWARE FOR PCR PRIMER'S
SELECTION/ANALYSIS "PRIMER-MASTER 1.0"
USER'S MANUAL
The program
"PRIMER-MASTER 1.0" is designated for search and selection
of best primers
and formation of optimal primer's
pairs for PCR
performance. The
primers selected by program
maximally satisfy to
various requirements,
providing high specific and
efficient ampli-
fication, and may be
used in broad range of application of PCR.
The program has
two main variants
of work :
"Primer_test" and
"Auto_search",
which are presented in main menu
bar as two first
items. The third
item "Utilities" contains three little subprograms.
The first two of them
have no independent significance but may prove to
be useful for work in
main variants. The last subprogram "New sequence
type" allows to
type-in and edit comfortably new nucleotide sequences.
To enter some item of menu or option,
press "Enter"-key, to leave
- press
"Escape". To pass from
one item or
option to other,
use
"Arrow" -
keys. To stop the work
of program, press "Pause". To
interrupt the work,
press any key and answer in
positive way to
proposed
questions. To leave the program
before start or
after
interruption, resort
to QUIT option of the main menu or press Escape.
<<<<<<<<<<<<<<<<<
<< AUTO_SEARCH <<
<<<<<<<<<<<<<<<<<
"Auto_search" implies
automatical search and selection of
best
primers and formation
of optimal primer's pairs for PCR. Working
in
this variant
program enables to
treat one or
several nucleotide
sequences and
to select primers,
which are universal
for all
considered sequences
(regime Universal) or, on the contrary,
specific
for only one out of
all sequences (regime Specific).
SEQUENCES
Option
"Sequences" allows to point out the names of being
introduced
sequence-files.This
option opens three active windows:
1)Upper string,
indicating the path to current
directory and file's
pattern;
2)Left window,
presenting files of current
directory according to
chosen pattern;
3) Right window,
displaying chosen and introduced file-names.
The user may move
along windows by means of Tab-key (or Tab+Shift - in
the opposite
direction). Being in Upper string he may change path and
file's pattern. Being
in the left window he can
move along files,
change directory or
drive (the last - by F2-key) and input
file-names
by means of pressing
Enter, Space-bar or Insert keys. User can
also
view any file by means
of F3-key or erase any file from disk
by means
of F8-key. Being in
the right window he can delete file-names (Delete-
key) and point
position for input of the next file. When all sequence-
files are introduced -
press F10. If You have changed Your mind - press
Escape and all actions
carried out in this session will be
abolished.
The number of treated
sequences is up to 100 in this version of program.
The length of any
sequence is limited by about 65000 nucleotides. The
total length of
introduced sequences is not limited. The maximal size
of viewable file is
about 300K.
SETTINGS
Option
"Settings" contains directives essential for work of the
program. These
directives may be changed by user.
1) "Output file names" - allows
to define the names of all output-
files which may be
formed if program works in variant "Auto_search".
The first output file contains optimal pairs
of primers. If no name
is defined by user,
the name "pairs."with
extension corresponding to
number of program's
start will be assigned (for example "pairs.025").
The second file ( by default the name is
"primers.XXX") contains the
best selected primers
without pair's formation.
The third file ( by default the name is
"primall.XXX") involves all
primers and some
information about them. This file
is formed only
according to user's
wish. In some cases this file may be very
useful,
especially for its
employment in "Primer_ test"
- so You can make
primer's design
actually in manual mode. The file may be
very large,
so before assigning
its formation please check availability enough space
on Your disk.
The last output file (by default the name is
"location.XXX") contains
localization of all
selected primers on
all considered sequences.
Obviously this file
will be formed only during the search of universal
primers for several
sequences (regime Universal).
Note, if particular
path for first output file is defined
by user,
this path will be
valid for all other output files. The extension of all
files equals the
extension of file "pairs."
-----------------------------------------------------------------------
2) "Number of parts" - number of
parts the sequence is divided in.
The division of
sequences in some parts allows to avoid situation when
all primers for plus
and minus chain are found in the same site and no
pairs can be formed. After
division the best primers will
be being
searched within
every part. It
provides pair's formation
and
considerably
accelerates program's work. The number of
parts may be
from 1 (the whole
sequence) to 9.
3) "Set of margin" - allows
to set
left ( 5') and right(
3')
boundaries on sequence
and to restrict the fragment where search
of
primers will be carried out. The restricted by
boundaries fragment
may be divided in some
parts as it has been described above.
4) "Sites for search" - allows to
point the boundaries of up
to
20 sites of sequence,
where primers are searched within. The defined
sites may not be
divided in parts.
5) "Amplify zone" - allows to
select primers which will amplify certain
fragment of certain
sequence (if working regime is Specific) or
amplify
respective fragments
of all proposed sequences (if working
regime is
Universal). In any
case user must point the left (5') and
right ( 3')
boundaries of
interesting fragment on the first sequence, and the best
primers will be being
searched within close zones ( 100 nucleotides
)
up- and down- stream
of the fragment.
The last three items
restrict in some way zones for primers
search and
selection and any of
them excludes all others.
------------------------------------------------------------------------
6) "Length of primer" - the
user may set
minimal and maximal
permissible length of
primers (in range from 16 to 26 nucleotides).
During program's work
this value may be changed
automatically within
set limits in one or
other direction depending
on regime of
work
(Specific or
Universal) because it is reasonable to expect
more long
primer to be more
specific and more short primer to be more universal.
7) "Size of ampl. fragment" -
allows to set the desirable lower and
upper limit of size of
flanked by primer's pairs fragment.
8)
"Difference of tempr." - permissible difference between
annealing temperatures
of primers sharing one pair.
9) "Temperature limits" - allows
to set permissible lower and upper
limits for
annealing temperature of
primers. Temperatures are
determined by the
nearest-neighbor method with taking into account the
nucleation constant.
Temperatures are calculated for
PCR buffers of
0.05 M concentration
of monovalent kationes. But in range from 0.025 M
to 0.075 M these temperatures are also valid.
10) "Save settings" -
preservation of majority of directives, which
have been set in
SETTINGS. If the directives are
not saved, their
values will be valid
only during current work of program.
As mentioned above automatical search
and selection of
best
primers may be carried
out in two regimes: "Specific" and "Universal".
SPECIFIC
Regime Specific allows to obtain
pairs of primers,
providing
specific amplification
of fragments of only one nucleotide
sequence
out of all proposed
ones.
It must be noted, the
name of sequence specific primers
are searched
for ( treated sequence
), shall be pointed out as the first in
option
"Sequences".
All primers will be tested in respect of their capability
of binding with
non-specific places (false priming) on treated sequence
and on all other
sequences, which names have been introduced after the
name of treated
sequence.
"Dir. for spec.check" - the user
may also define targets for check
of primers specificity
by means of pointing the directory containing
these sequence-files.
"Auto_margin" (switch On -
Off by
Enter key) -
this regime is
foreseen for the
search of specific primers for one out of related and
probably
sequenced from different
position sequences. It allows to
align sequences
approximately and to avoid choice of
primers to sites
of treated sequence,
which are not presented in other ones.
UNIVERSAL
Regime "Universal" allows to
obtain pairs of primers, which are
universal for all
considered sequences.
Names of sequences
must be pointed in option "Sequences" and the order
of introduction of
names does not matter.
Using "Sequences for spec.check",
up to 100 additional sequences,
which amplification is
undesirable, may be introduced. The same may be
done by using option
"Directory for spec.check" (the user
must point
only directory
containing unlimited number of such sequences). In both
cases the program
works so that these sequences can not
be amplified
in PCR with primers
universal for main group of sequences. To cancel
settings made in
"Sequences for spec.check" - enter
this option and
remove all file-names
from right window or only press Escape.To cancel
settings made in
"Directory for spec.check" - enter
this option and
press Escape. Since
these two options exclude one other,
activity in
any of them abolishes
settings made in other option.
Option "Difference of size" sets
an maximal difference between the
lengths of fragments
of different sequences, amplified by
the same
pair of primers.
========================================================================
<<<<<<<<<<<<<<<<<
<< PRIMER_TEST <<
<<<<<<<<<<<<<<<<<
"Primer_test" - allows testing
your oligonucleotide with respect
to any sequence (using
option "Test on sequences") and/or to some
intrinsic properties
of the
oligonucleotide it-self (using
option
"Self-test").
Option "Primer" allows to
handle the oligonucleotides to be tested.
The user may type-in
the sequence of oligonucleotide to be tested. Besides
the common symbols A,
G, C and T, the ambiguous bases may be included,
according to the table
belong:
--------------------
| Symbol | Meaning |
--------------------
| B
| not A |
| D
| not C |
| H
| not G |
| K
| G or T |
| M
| A or C |
| R
| A or G |
| S
| C or G |
| V
| not T |
| Y
| C or T |
| W
| A or T |
| N
| unknown |- the nucleotide wittingly non-complementary and
| | non-homology to any nucleotide of sequence
| X
| A C G T |- the nucleotide wittingly complementary or
|__________________| homology to any nucleotide of sequence
The user may create
the list of from 1 to 50 oligonucleotides.
Active keys:
Enter - fixes the typed in working
window sequence as the being
tested oligonucleotide
under current number (shown in the
left upper
corner of window);
F9 - stores the typed in working
window sequence at the end
of
created list;
F8 - removes the shown in working
window sequence from the list;
F5 - allows to write the shown in working
window sequence in file
The use must define the
path and name
of file (by
default
"primlib.dat" in current
directory) and press Enter. Then he can
type a short comment to stored
oligonucleotide and press Enter or
only
press Enter (then
the primer will
be stored without
comments). If despite pressing Enter the
user will press Escape,
the oligonucleotide will not be
written in file. If the
file
where the oligonucleotide is being stored
already exist, the new
oligonucleotide will be added at the end
of file );
Up and Down arrows - allows moving along
the list;
Alt+R - allows to reorientate the 5' and 3'
termini of oligonucleotide;
Alt+C - allows to make the typed-in
oligonucleotide complementary;
Escape - closes the working window
and abolishes all changes in
working window if they
have not been fixed by Enter of F9.
Sequence of being tested
oligonucleotide(s) can be input in one
more way : being in
the option "Sequences" You can find file of Your
interest and view it
by F3-key. After pressing
F7 and cursor's
occurring You can mark
any number of symbols (up to 60 -
the maximal
length of tested
oligonucleotide). Marking and
unmarking may be
performed by
Shift+Arrow-keys. Only marked
letters are taken to be
the tested
oligonucleotide. Direction of
marking coincides with
direction of
oligonucleotide: the first marked symbol corresponds to
5' terminus
and the last
marked symbol -
to 3' terminus. To
input marked
string as the
oligonucleotide - press
F10 (the
oligonucleotide will
be input under current number -
the number You
could see having
opened option "Primer"). If you want
to create the
list - press F9 (the
oligonucleotide will be stored at
the end of
list). If You want
marked string to be input in complementary way -
press Shift+F10 or
Shift+F9 respectively. To cancel all You have marked
- press F7. To leave
viewer - press Escape.
You can see
the text
of input oligonucleotide(s) in option
"Primer", where You can edit it.
If You did any changes
in oligonucleotide's sequence - press Enter.
Option "Sequences" allows to
input sequences, on which the user
wishes to test
oligonucleotide(s). This option completely
corresponds
to the same option in
AUTO_SEARCH and all its operations are described
above.
Alternatively, option
"Directory" allows to point
path, name and
file's pattern of
directory containing any number of sequence-files on
which testing of
oligonucleotide is desirable.
Option "Test on sequences"
enables to test oligonucleotide with
respect to sequences
pointed either in option "Sequences" or in option
"Directory".
It opens three sub-options.
1) Sub-option "Best energy" allows
to set the number of
most
preferable in
energetic terms sites of sequences for binding of tested
primer. To do it,
enter the option (by Enter) press Enter
again and,
after cursor's
occurring, type the number. Search
of set number of
sites may be performed
on every sequence or on all
sequences on the
whole (switch
"all" - "every" by Enter-key).
2) Sub-option "Complementarity"
allows to find sites of sequences
where tested oligonucleotide has level of complementarity
equal or
exceeding the
threshold set by user. The search may be performed in
three regimes:
a) when "Total Complementarity"
> 0 and "3'end Complementarity" = 0 -
search of sites of
sequences having level of complementarity equal or
exceeding threshold
set for "Total Complementarity";
b) when "Total Complementarity" =
0 and "3'end Complementarity" > 0 -
search of sites of
sequences having sufficient continuous complementa-
rity with 3' termini
of tested oligonucleotide
independent on total
complementarity.
c) if "Total Complementarity" >
0 and "3'end Complementarity" > 0 -
search of sites
of sequences corresponding to both set levels in
combination.
The search may be
carried out on both chains of sequences or
only on
plus or minus chain
(switch by Enter key).
3) Sub-option "Homology search"
is very similar to previous option
but the main
criterion here is
homology of oligonucleotide and
sequence. Homology
search may be performed in three regimes:
a) when "Total Homology" > 0
and "Non-stop Homology" = 0 - search of
sites of sequences
having level of homology equal or exceeding
threshold set for
"Total homology";
b) when "Total Homology" = 0 and
"Non-stop Homology" > 0 - search of
sites of
sequences having sufficient
continuous homology with
tested oligonucleotide
independent on total homology.
c) if "Total Homology" > 0 and
"Non-stop Homology" > 0 -
search of
sites of sequences
corresponding to both set levels in combination.
Items "Max.gap in primer" and
"Max.gap in seq." allows to set the
maximal permissible
size of gaps in primer/sequence (from 0 to 9) which
is taken into account
during the homology search.
Like it is in previous
sub-option, search of homology may be performed
on both chains of
sequences or only on plus or minus chain.
Option
"Self-test"
allows to obtain
information about some
properties of tested
oligonucleotide it-self. Namely,
information
about melting
temperature molecular weight and possibility of stem-loop
structure formation is
accessible. To obtain this information in output
file user must mark
corresponding item inside "Self-test" (mark-unmark
by Enter-key). The
length, annealing temperature and mol. weight of oli-
gonucleotide is also
displayed in the active window of option "Primer".
Note, only actually
probable hairpins will be displayed in
output-file.
Option "Output file name"
allows to define the name and/or path
for output file. By
default the name "primtest.XXX" with
extension
corresponding to the
number of program's start is assigned.
Note, all possible or
only one setting for test has been done
- it
does not matter.
To start testing
oligonucleotide(s) the user must open option
"Primer"
and fix in the working
window the primer which user wants to test as the
first. Then the user
must close the window and place the cursor on the
"Run".
Sequential pressing Enter key will be followed by
testing
oligonucleotide(s)
from the list one after other.
========================================================================
<<<<<<<<<<<<<<<
<< UTILITIES <<
<<<<<<<<<<<<<<<
This item of main menu involves three
sub-programs:
"Output file
treatment", "Complementary chain" and "New sequence
type".
"Output file treatment" allows
to form, on the base of existing
output files new pairs
of primers corresponding to newly set
tempera-
tures difference
and/or permissible length of flanked
fragments. The
user may also set the
desirable number of pairs. The item
"Difference
of size"
corresponds to similar option in "Universal" of
"Auto_search"
and sets the maximal
difference between the lengths of fragments
of
different sequences
amplified by the same primer's pair. The files
in
format of
"primers.XXX" or "pairs.XXX" may be subject for treatment.
"Complementary chain" is
designated for building "minus"-chain to
existing
"plus"- chain of nucleotide sequence. You have to point name
of file containing
sequence you are interested in, and
name of file
for converted
sequence.
"New sequence type" is
designated to facilitate typing-in new
nucleotide sequence
parallelly and comparing it to related sequence-
pattern.
Option
"Redesignate keys" allows to redesignate seven active keys denoting
nucleotides
(g,a,t,c,n) and deletion ("-" symbol in newly typed sequence)
and insertion
("-" symbol in pattern sequence opposite the inserted
nucleotide in new
sequence). Note, do not mix up insertion and deletion
symbols with standard
Delete and Insert keys of keyboard. To redesignate
some key press Enter
and after cursor occurence press any key you wish
to denote respective
symbol.
Using the
"Pattern sequence" option one can select the sequence-file which
will be used as
reference pattern for typing new sequence.
When the keys are comfortably redesignated
and the pattern sequence is
selected one should
Run the program. It takes and displays the pattern
sequence file so that
the header part of file is presented without changes
but the sequence
itself is presented as 60 nucleotide length lines (passive
lines) alternated with
empty lines where new sequence may be typed (active
lines). The cursor can
be placed only on active line. If the typed-in
symbol mismatches the
respective nucleotide in passive line, the sound
signal will occur and
this new nucleotide will be displayed as capital red
letter. In general,
all mismatches are displayed as capital red letters.
Besides the
redesignated keys the following ones are active:
Insert key - turns on/off the insertion mode of editor
(by default the
mode is "off");
Space-bar - input space symbol;
Delete & BackSpace
- have standard for all text editors functions;
Ctrl+BackSpace -
removes the insertion symbol from the passive line (if any)
together with respective
"inserted" symbol in active line.
If no "-" symbol is
in passive line Ctrl+BackSpace works as
BackSpace.
F2 key - allows to write both sequences
(pattern and newly typed) in
file. This file will be
recognized by program and typing new
sequence may be carried on
again.
Shift+F2 - allows to write only newly typed
sequence in file in EMBL
gene bank format.
F7 key - allows the search for short motifs. The
user should type
the short (up to 60 nucl.)
sequence for search and press
Enter. The 3' and 5' termini
of typed-in sequence may be
reorientated by Alt+R keys or
this sequence may be obtained
in complementary form by
Alt+C keys. The 100 position with
most high homology will be stored
in memory and the best
one will be displayed. To see
all other positions user shall
press Ctrl+Right Arrow
(moving in direction of less homology)
or Ctrl+Left Arrow. The
homology level is determined taking
into account the possibility
of 1-2 nucleotide
deletions/insertions. The
search for short motifs may be
performed on sequences of no
more than 50000 nucleotides
length (this value is valid
if the header part of sequence-file
except the last line
containing "SQ" or "ORI..." is removed).
F12 key - allows to switch the direction of
typing from the normal
left-to-right to the
right-to-left direction and vise versa.
Ctrl+any key denoting
the nucleotide - inputs the insertion symbol in passive
line and the
"inserted" nucleotide in active line at the
same time.
Escape - quit the program.
================================================================================
THE FORMAT OF SEQUENCE FILES
The only requirement to sequence file
format is as follows:
The line preceding the
first nucleotide line of file must begin from
the combination of
capital letters "SQ" or "ORI" or "ORIGIN". If the
file
you wish to handle as
sequence file breaks this rule, please add necessary
letters in respective
place.
OUTPUT FILES
The file "primers.XXX" contains
all selected primers
without
pair's formation. This
file is preferable for "Output file treatment",
because some primers
can not form pairs and therefore
are absent in
file
"pairs.XXX".
In the left column of
files "pairs.XXX", "primers.XXX", "location.XXX"
information about
sense primers ( marked as (+)) is situated,
in the
right column -
information about anti-sense primers ( marked as (-)).
If program works in
variant UNIVERSAL the files "primers" and "pairs"
contain figures in
brackets in line "Annealing temperature". These
figures mean the
annealing temperature of
primer connected with
possible partial
uncomplementarity of primer in target
place. This
temperature shall be
used during the first 4-5 cycles
of PCR. The
following cycles shall
be performed at the temperature pointed without
brackets. If the
figures in brackets and without any are the
same it
means that the primer
has complete complementarity in target places on
all sequences.
Some primers obtained after work of
SPECIFIC may be supplied by
special mark
"*". They have high probability of
nonspecific binding,
but are unable to
be extended in places of
this binding due
to
uncomplementarity of
their 3'- termini
and absence of
3'-5'-
exonuclease activity
of thermostable polymerases used
in PCR. These
primers will be
selected only if there are no better ones.
TERMINOLOGY
MAX.ENERGY ( Gm) - the binding energy (
exactly the standard free
energy of Gibbs) of
primer and template in specific place. If program
works in regime
"Universal", the mean value of energies of binding of
primer with respective
sites of all considered sequences is hadeled as
MAX.ENERGY (maximum)
DELTA ( Gm - Gs) - difference between
binding energies of primer and
template in specific
and most profitable non-specific place (difference
between energetic
maximum and submaximum).
GENERAL INFORMATION AND PRINCIPLES
There are some main features, which
distinguish the presented
software from other
programs for primer's choosing.
1) First of all, it enables to treat
several nucleotide sequences
and to
select primers, which
are universal for
all considered
sequences or, on the
contrary, specific for
only one out
of all
sequences. This
capability of the program allows it to be useful, for
example, for the
development of PCR-based
diagnostic systems. The
algorithm
exploited in program allows to
reveal conservative and
variable sites of
sequences without alignment's building.
The method
is very reliable and quite rapid. It lifts
principal limitations from
the number of treated sequences and allows finding specific primers
for one out of several very similar sequences or universal for very
unlike sequences.
2)
The program pays particular attention to the
evaluation of
specificity of selected primers. Selected
primer must be able of
reliable binding and extension in specific
place of proper sequence
(if working regime is "Specific")
or in
respective sites of
all
considered sequences (if regime is
"Universal") and have
the least
probability of primer-template binding and
extension in any other
place. To estimate the reliability of
specific and probability of
non-specific interactions the energetic
criterion is proposed. It is
based on assumption that certain primer
interacts with template in
random way, then the probability and
reliability of primer-template
binding is primarily defined by
thermodynamic stability of
duplex
and most perfectly described by the standard
free energy of Gibbs
(dG).
So, the binding energy of primer
and template is
estimated, by
nearest-neighbor method according to the
principle
"all-or-none".
The binding energy of primer estimated in
its specific place
is
taken as an energetic maximum. If regime
is "Universal", the
mean
binding energy of primer in its specific sites
of all considered
sequences is taken to be the
energetic maximum. Such
approach
enables to take into account both the
destabilizing influence of
primer's partial uncomplementarity in its
specific site and
the
probability of this uncomplementarity.
The energy is also estimated for any
other places of
possible
extension of this primer (places
where 3'termini nucleotides
of
primer
are complementary to
sequence), for any
possible
conformations of it, including formation
of internal loops, bulge
loops, asymmetric loops and dangling
ends. The permitted size of
bulge loop on the primer's side is up to 2
nucleotides, on the side
of sequence - 1 nucleotide. The maximal value
of binding energy in
any nonspecific place is taken as energetic
submaximum. The primers
having most high energetic maximum and the
most difference between
maximum and submaximum are selected as the
best ones. They form most
stable
bond with template
in specific site
and have minimal
probability of nonspecific interaction at certain
conditions. The
application of energetic criterions, first
of all evaluation of
submaximum, guarantees principally more
high accuracy of primer's
specificity estimation. Such accuracy may not
be provided by usually
used for this goal criterion of
complementarity.
It
must be noted that the data concerning
DNA-duplex energies
and conditions of helix to coil
transition are very
contradictory
and unsufficient. Moreover the conventional
principle "all-or-none"
describes such a transition not perfectly
enough. Nevertheless,
estimation of all primers in the same
manner makes this draw-back
rather negligible and enables to select the
really best primers.
3)
The program allows to carry out the search and selection of
primers within the interesting sites of
sequences, or to carry out
selection of primers and formation of primer's
pairs, which provide
amplification of interesting fragment of
sequences. It may be very
useful for the selections of primers for site-directed mutagenesis
or for preparation for cloning or sequencing.
4)
The program has a very important capability to adapt itself to
the treated sequences. A lot of criterions
of selection and
other
values may be softened automatically, if no
primer is selected or no
pairs are formed using the most stringent
forms of these criterions.
You will obtain " 0 pairs are
formed " and void
output-files only
when virtually all possibilities are depleted.
Of course, the altera-
tion of criterions proceeds only within the
reasonable limits.
Besides to be high specific, selected primers have also some
properties:
a) they contain no sequence repeat structure;
b) they contain no large GC-rich sites and no
stretched repeats of the
same nucleotides;
c) they are free of hairpins (the annealing
temperature of primer must
be at least 25 C higher than melting
temperature of most stable hairpin,
which could be formed by this primer);
d) they can not form any dimmer structures;
e) their annealing temperature is within
limits set by user ( but not
lower than 35 C and not higher than 73 C ).
The particular attention is paid to the
3'-ends of primers, since just
their
properties determine the
possibility or impossibility of
primer's extension. For example, the universal
primers must have at
least three complementary nucleotides at their
3'-terminies in all
target places on all sequences. On the other
hand, during the search of
energetic submaximum ( place of most
profitable nonspecific binding ),
all sites, where primer has complementarity of
even two 3' nucleotides
are considered as the sites of
possible primer's extension.
This
criterion may seem to be too stringent, but
we suppose that
it is
better to overdo in this term, than to
overlook potentially dangerous
site.
Requirements for pair's formation:
1) Difference between annealing temperatures
of both primers is no
more than value set by user;
2) The size of fragments flanked by primers is
within limits set by
user;
3) Primers can not bind with each other (the
maximal possible energy
of their binding must not exceed the
submaximum of each primers);
4) 3'-end of one primer can not bind with any
site of other one;
5) No one primer forms hairpin at the
annealing temperature of second
primer in pair;
6) The length of fragments of different
sequences amplified by univer-
sal primers do not differ from each other more
then by value set by
user.
The presented software is developed on Turbo-C language and may be
used on every IBM-compatible computer.