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| #!/usr/bin/perl
use warnings;
use strict;
use Tk;
my $main = MainWindow->new(
-background => 'blue',
-title => "Calcul de la température de fusion",
);
$main->minsize( 250, 150 );
# lien : http://www.omrf.org/omrf/research/09/FrankLab/HYBNOTES.html
my $RefLabel = $main->Label(
-text => "T° basée sur l'analyse des plus proches voisins (revu par Genosys) ",
-background => 'blue',
-foreground => 'white',
-justify => 'left',
);
my $SeqLabel = $main->Label(
-text => 'Séquence : ',
-background => 'blue',
-foreground => 'white',
-justify => 'left',
);
my $SeqEntry = $main->Entry(
-background => 'white',
-width => 50,
);
my $NaLabel = $main->Label(
-text => 'Concentration en sel (M): ',
-background => 'blue',
-foreground => 'white',
);
my $NaEntry = $main->Entry(
-background => 'white',
-textvariable => '5E-02',
-width => 15
);
my $CtLabel = $main->Label(
-text => 'Concentration en amorces (M): ',
-background => 'blue',
-foreground => 'white',
);
my $CtEntry = $main->Entry(
-background => 'white',
-textvariable => '2E-07',
-width => 15
);
# bouton initialiser
my $btn_initialiser = $main->Button(
-text => 'Initialiser',
-activeforeground => 'red',
-borderwidth => 5,
-command => \&Initialiser,
);
# bouton aide
my $btn_aide = $main->Button(
-text => 'Aide',
-activeforeground => 'red',
-borderwidth => 5,
-command => [ \&Aide, $main, ],
);
# bouton calculer
my $btn_tm = $main->Button(
-text => 'Tm',
-activeforeground => 'red',
-borderwidth => 5,
-command => [ \&Result, $main, ],
);
# bouton quitter
my $btn_quitter = $main->Button(
-text => 'Quitter',
-activeforeground => 'red',
-borderwidth => 5,
-command => \&Quitter,
);
# Effet clavier, touche entrée
$main->bind( 'Tk::Entry','<Return>', sub { $btn_tm->invoke(); } );
# Placement de mes widgets
$RefLabel->grid( -row => 0, -column => 0, -sticky => 'w', -pady => 20, -columnspan =>2);
$SeqLabel->grid( -row => 1, -column => 0, -sticky => 'w' );
$SeqEntry->grid( -row => 1, -column => 1, -sticky => 'w', -columnspan => 2);
$NaLabel->grid( -row => 2, -column => 0, -sticky => 'w' );
$NaEntry->grid( -row => 2, -column => 1, -sticky => 'w', -columnspan => 2);
$CtLabel->grid( -row => 3, -column => 0, -sticky => 'w' );
$CtEntry->grid( -row => 3, -column => 1, -sticky => 'w', -columnspan => 2);
$btn_initialiser->grid( -row => 4, -column => 0, -pady => 20, );
$btn_tm->grid( -row => 4, -column => 1, -pady => 20 );
$btn_aide->grid( -row => 4, -column => 2, -pady => 20 );
$btn_quitter->grid( -row => 4, -column => 3, -pady => 20 );
MainLoop;
# Fonctions utiliser par l'interface graphique
sub Initialiser {
$SeqEntry->delete(0, 'end');
$NaEntry->delete(0, 'end');
$NaEntry->insert('end', '5E-02');
$CtEntry->delete(0, 'end');
$CtEntry->insert('end', '2E-07');
return;
}
sub Quitter {
exit(0);
}
# Aide
sub Aide {
my ($fenetre_principale) = @_;
my $help_frame = $fenetre_principale->Toplevel(
-title => 'AIDE',
-background => 'blue',
);
$help_frame->minsize( 1200, 700 );
$help_frame->Label(
-text => 'HYBRIDIZATION NOTES - Melting and Annealing Temperature',
-foreground => 'white',
-background => 'blue',
)->pack(qw/ -side top -fill x/);
$help_frame->Button(
-text => 'Fermer',
-command => sub { $help_frame->destroy; },
)->pack(qw/ -side bottom /);
my $help_text
= $help_frame->Scrolled( 'Text', -scrollbars => 'osoe', )->pack(qw/ -fill both -expand 1 /);
$help_text->insert( 'end',
q{ SITE DE REFERENCE : http://www.omrf.org/omrf/research/09/FrankLab/HYBNOTES.html
AUTEUR : Frank, M. B. Hybridization Notes - Melting and Annealing Temperature. In: Frank, M. B. ed. Molecular Biology Protocols. 1997. Oklahoma City. Revision Date: October 2, 1997.
Other estimates of melting points have been determined for DNA3 or RNA4 based on nearest neighbor analysis (reviewed by Genosys5). Breslauer, et al3 showed that melting behavior of a DNA duplex is predictable from its primary sequence.
Tm = 1000(DH)/[A+DS)+Rln(Ct/4)]-273.15+16.6log(Na+)].
where
DH = the sum of nearest neighbor enthalpy changes moving one base at a time through the sequence
A = correction for initiation of pairing (= -10.8)
DS = the sum of nearest neighbor entropy changes
R = 1.987 cal deg-1 mol-1)
Ct is the molar concentration of strands.
For self-complementary strands, the term "Ct/4" is replaced by Ct.
The values for DH and DS are shown in the table.
Nearest Neighbor DH DNA (kcal/mol) DH RNA (kcal/mol) DS DNA (cal/mol); DNA DS RNA (cal/mol)
AA or TT - 9.1 - 6.6 -24.0 -18.4
AT - 8.6 - 5.7 -23.9 -15.5
TA - 6.0 - 8.1 -16.9 -22.6
CA or TG - 5.8 -10.5 -12.9 -27.8
GT or AC - 6.5 -10.2 -17.3 -26.2
CT or AG - 7.8 - 7.6 -20.8 -19.2
GA or TC - 5.6 -13.3 -13.5 -35.5
CG -11.9 - 8.0 -27.8 -19.4
GC -11.1 -14.2 -26.7 -34.9
GG -11.0 -12.2 -26.6 -29.7
As an example, a 1 µM solution of the probe mentioned above (AGGTCATTG) in a 150 mM solution has a predicted Tm of:
Tm = 1000(-7.8-11.0-6.5-5.6-5.8-8.6-9.1-5.8) / [-10.8+(-20.8-26.6-17.3—13.5-12.9-23.9-24.0-12.9) + 1.987ln[(1E-06)/4]] - 273.15 + 16.6log(0.15)
= [-60900 / (-10.8-151.9-30.2)] - 273.15 - 13.7
= (-60900/-192.9) - 286.9
= 29°C
Calcul de la temperature optimale de fusion du duplex amorce-ADN
en utilisant des criteres energetiques
dH,dS de la formation de l'helice
R cste gaz parfait= 1.987 cal*k-1.mol-1
C concentration de l'oligo en Molaire
Na+ concentration en sels en Molaire
dH
Tmam = _________________ -273.15 + 16.6 log[Na+] (1),(2),(3)
dS + R*ln(C/4)
Parametres thermodynamiques des
plus proches voisins pour l ADN (1):
______________________________________
Interaction dH dS dG
______________________________________
AA/TT 9.1 24.0 1.9
AT 8.6 23.9 1.5
TA 6.0 16.9 0.9
CA/TG 5.8 12.9 1.9
GT/AC 6.5 17.3 1.3
CT/AG 7.8 20.8 1.6
GA/TC 5.6 13.5 1.6
CG 11.9 27.8 3.6
GC 11.1 26.7 3.1
GG/CC 11.0 26.6 3.1
initiation 10.8
______________________________________
dG, dH Kcal/mol
dS cal/mol (1 cal = 4.184 J)
______________________________________
},
);
return;
}
# calcul du Tm
sub Result {
my ($fenetre_principale) = @_;
# récupération des données
#----------------------------
my $seq = $SeqEntry->get();
my $Na = $NaEntry->get();
my $Ct = $CtEntry->get();
# suppression des espaces éventuels
#------------------------------------
$seq =~ s/\s//g;
$seq = uc($seq);
# Vérification
#---------------
if ( $seq !~ m/^[ATCGRKSWMYDVBHN]+$/ ) {
# fenêtre d'erreur
#--------------------
my $error_frame = $fenetre_principale->Toplevel(
-title => 'MESSAGE D\'ERREUR',
-background => 'red',
);
$error_frame->minsize( 250, 50 );
$error_frame->Label(
-text =>
"La séquence ne peut contenir\nque des lettres du code IUPAC",
-background => 'red',
-font => "courrier 10 bold",
-foreground => 'black',
)->pack;
}
else {
#-------------------------
# fenêtre de résultat
#-------------------------
my $amorce_l = length($seq);
my %correspondances =
(
'R' => ['A', 'G'],
'K' => ['G', 'T'],
'S' => ['C', 'G'],
'W' => ['A', 'T'],
'M' => ['A', 'C'],
'Y' => ['C', 'T'],
'D' => ['A', 'G', 'T'],
'V' => ['A', 'C', 'G'],
'B' => ['C', 'G', 'T'],
'H' => ['A', 'C', 'T'],
'N' => ['A', 'C', 'G', 'T']
);
my @liste_amorces;
core( sub { push (@liste_amorces, $_);}, $seq) ;
my @a_tm;
my %h_tm;
foreach my $seq (@liste_amorces){
my $tm = Tm($seq,$Ct,$Na);
$h_tm{$seq}= $tm;
push (@a_tm, $tm);
}
my $moyenne_tm;
map{$moyenne_tm += $_} @a_tm;
$moyenne_tm /= int(@a_tm);
$moyenne_tm = sprintf("%.3f", $moyenne_tm);
my $fenetre_resultat = $fenetre_principale->Toplevel(
-title => "Tm de $seq (Na : $Na Ct : $Ct)",
-background => 'blue',
);
$fenetre_resultat->minsize( 500, 600 );
$fenetre_resultat->Label(
-text => 'Résultat',
-foreground => 'white',
-background => 'blue',
)->pack(qw/ -side top -fill x/);
$fenetre_resultat->Button(
-text => 'Fermer',
-command => sub { $fenetre_resultat->destroy; },
)->pack(qw/ -side bottom /);
my $text_resultat
= $fenetre_resultat->Scrolled( 'Text', -scrollbars => 'osoe', )->pack(qw/ -fill both -expand 1 /);
# Tag GRAS
$text_resultat->tagConfigure('GRAS',
-font => '{Times New Roman} 8 {bold}',
);
# Tag Rouge
$text_resultat->tagConfigure('ROUGE',
-foreground => 'red',
);
# lignes d'affichage
while ( my ( $s, $t ) = each %h_tm ) {
$text_resultat->insert( 'end', "$s : $t °C\n" );
}
if ( int(@a_tm) > 1 ) {
$text_resultat->insert( 'end', "$seq : $moyenne_tm °C\n", ['GRAS','ROUGE'] );
}
}
return;
}
sub core
{
my ($code, $amorce) = @_ ;
my %correspondances = (
'R' => ['A', 'G'],
'K' => ['G', 'T'],
'S' => ['C', 'G'],
'W' => ['A', 'T'],
'M' => ['A', 'C'],
'Y' => ['C', 'T'],
'D' => ['A', 'G', 'T'],
'V' => ['A', 'C', 'G'],
'B' => ['C', 'G', 'T'],
'H' => ['A', 'C', 'T'],
'N' => ['A', 'C', 'G', 'T']
);
if ( my ($start, $middle, $end) = ($amorce =~ /^([ACGT]*)([RKSWMYDVBHN])(\w*)$/i) ) {
foreach my $alt (@{$correspondances{$middle}}) {
core( $code, "$start$alt$end" );
}
}
else {
local $_ = $amorce ; # Le mot clé "local" "localise" une variable globale de sorte que à la sortie du bloc celle-ci reprenne la valeur qu'elle avait juste avant d'être localisée
$code->(); # $code->() est simplement une manière d'appeler la fonction référencée par $code
}
}
sub Tm{
my $Amorce = $_[0];
my $Ct = $_[1];
my $Na = $_[2];
my %dH =
(
AA => -9.1,
TT => -9.1,
AT => -8.6,
TA => -6.0,
CA => -5.8,
TG => -5.8,
GT => -6.5,
AC => -6.5,
CT => -7.8,
AG => -7.8,
GA => -5.6,
TC => -5.6,
CG => -11.9,
GC => -11.1,
GG => -11.0,
CC => -11.0,
);
my %dS =
(
AA => -24.0,
TT => -24.0,
AT => -23.9,
TA => -16.9,
CA => -12.9,
TG => -12.9,
GT => -17.3,
AC => -17.3,
CT => -20.8,
AG => -20.8,
GA => -13.5,
TC => -13.5,
CG => -27.8,
GC => -26.7,
GG => -26.6,
CC => -26.6,
);
my $H = 0;
my $S = 0;
my $NbScan = length($Amorce)-2+1;
for(my$i=0; $i<$NbScan; $i++)
{
my $N2 = substr($Amorce, $i, 2);
if((!exists $dH{$N2}) | (!exists $dS{$N2})){print "ERREUR N2 ($N2) $Amorce, $i\n";};
unless($N2=~/N/i)
{
$H += $dH{$N2};
$S += $dS{$N2};
}
}
my $Tm = 1000 * $H / ((-10.8 + $S)+1.987*log($Ct/4)) - 273.15 + 16.6*(log($Na)/log(10));
$Tm = sprintf("%.3f", $Tm);
return $Tm;
} |
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