#
# GeneDesign module for restriction enzyme handing
#
=head1 NAME
Bio::GeneDesign::RestrictionEnzyme
=head1 VERSION
Version 5.52
=head1 DESCRIPTION
GeneDesign object that represents a type II restriction enzyme
=head1 AUTHOR
Sarah Richardson <SMRichardson@lbl.gov>
=cut
package Bio::GeneDesign::RestrictionEnzyme;
use Bio::GeneDesign::Basic qw(:GD);
use Carp;
use strict;
use warnings;
use base qw(Bio::Root::Root);
our $VERSION = 5.52;
my $IIPreg = qr/ ([A-Z]*) \^ ([A-Z]*) /x;
my $IIAreg = qr/\A \w+ \(([\-]*\d+) \/ ([\-]*\d+)\)\Z /x;
my $IIBreg = qr/\A\(([\-]*\d+) \/ ([\-]*\d+)\) \w+ \(([\-]*\d+) \/ ([\-]*\d+)\)\Z /x;
my %RE_vendors = (
B => "Invitrogen", C => "Minotech", E => "Stratagene Agilent",
F => "Thermo Scientific Fermentas", I => "SibEnzyme", J => "Nippon Gene Co.",
K => "Takara", M => "Roche Applied Science", N => "New England Biolabs",
O => "Toyobo Technologies", Q => "Molecular Biology Resources",
R => "Promega", S => "Sigma Aldrich", U => "Bangalore Genei", V => "Vivantis",
X => "EURx", Y => "CinnaGen"
);
my %methtrans = (b => "blocked", blocked => "blocked",
i => "inhibited", inhibited => "inhibited",
u => "unknown", unknown => "unknown"
);
=head1 CONSTRUCTOR METHODS
=head2 new
You can create a new enzyme or clone an existing enzyme to create a new instance
of an abstract enzyme definition. To do this, provide the -enzyme flag; the
constructor will ignore every other argument except for -start.
Required arguments:
EITHER
-enzyme : a Bio::GeneDesign::RestrictionEnzyme object to clone
OR
-id : The name of the enzyme (i.e., BamHI)
-cutseq : The string describing the enzyme's recognition and cleavage
site
Optional arguments:
-temp : The incubation temperature for the enzyme
-tempin : The heat inactivation temperature for the enzyme
-score : A float score, usually the price of the enzyme in dollars
-methdam : Sensitivity to dam methylation; can take the values
b or blocked,
i or inhibited,
u or unknown,
if undefined, will take the value indifferent.
-methdcm : Sensitivity to dcm methylation; can take the values
b or blocked,
i or inhibited,
u or unknown,
if undefined, will take the value indifferent.
-methcpg : Sensitivity to cpg methylation; can take the values
b or blocked,
i or inhibited,
u or unknown,
if undefined, will take the value indifferent.
-vendors : a string of single letter codes that represent vendor
availability - no spaces. see vendor() for a list of the
codes.
-staract : Whether or not the enzyme exhibits star activity - 1 or 0.
-buffers : a hash reference; keys are buffer names and values are the
enzyme activity in that buffer. For example:
NEB1 => 50, NEB2 => 100, etc.
-start : An integer representing an offset; usually used only in
cloned instances, as opposed to abstract instances.
-exclude : An arrayref full of ids for enzymes that should be
considered mutually exclusive to this enzyme - see exclude()
=cut
sub new
{
my ($class, @args) = @_;
my $self = $class->SUPER::new(@args);
my ($object, $id, $cutseq, $temp, $tempin, $score, $methdam, $methdcm,
$methcpg, $vendors, $staract, $buffers, $start, $exclude, $aggress) =
$self->_rearrange([qw(ENZYME ID CUTSEQ TEMP TEMPIN SCORE METHDAM METHDCM
METHCPG VENDORS STARACT BUFFERS START EXCLUDE AGGRESS)], @args);
if ($object)
{
$self->throw("object of class " . ref($object) . " does not implement ".
"Bio::GeneDesign::RestrictionEnzyme.")
unless $object->isa("Bio::GeneDesign::RestrictionEnzyme");
$self = $object->clone();
}
else
{
$self->throw("No enzyme id defined") unless ($id);
$self->{'id'} = $id;
$self->throw("No cut sequence defined") unless ($cutseq);
$self->{'cutseq'} = $cutseq;
my $recseq = $cutseq;
$recseq =~ s/\W*\d*//xg;
$self->{'recseq'} = $recseq;
#Regular expression arrayref to use for enzyme searching
#Should store as compiled regexes instead
$self->{'regex'} = _regarr($recseq);
my $sitelen = length($recseq);
$self->{'length'} = $sitelen;
#Enzyme Class and Palindromy
my ($lef, $rig) = (q{}, q{});
if ($cutseq =~ $IIPreg)
{
$lef = length($1);
$rig = length($2);
$self->{'class'} = "IIP";
$self->{'classex'} = $IIPreg;
if ($lef == $rig)
{
$self->{'palindromy'} = "unknown";
}
else
{
my $inlef = $lef;
$inlef = length($recseq) - $inlef if ($inlef > (.5 * length($recseq)));
my $mattersbit = substr($recseq, $inlef, length($recseq) - (2 * $inlef));
if ($mattersbit && $mattersbit =~ $ambnt && length($mattersbit) % 2 == 0)
{
$self->{'palindromy'} = "pnon";
}
elsif ($mattersbit && $mattersbit eq _complement($mattersbit, 1))
{
$self->{'palindromy'} = "pal";
}
elsif ($mattersbit)
{
$self->{'palindromy'} = "nonpal";
}
}
}
elsif ($cutseq =~ $IIBreg)
{
$lef = int($1);
$rig = int($2);
$self->{'class'} = "IIB";
$self->{'classex'} = $IIBreg;
$self->{'palindromy'} = "pnon";
}
elsif ($cutseq =~ $IIAreg)
{
$lef = int($1);
$rig = int($2);
$self->{'class'} = "IIA";
$self->{'classex'} = $IIAreg;
$self->{'palindromy'} = "pnon";
}
else
{
$self->{'class'} = "unknown";
}
#Enzyme type
my $type;
if ($lef < $rig)
{
$type .= "5'";
$self->{'inside_cut'} = $lef;
$self->{'outside_cut'} = $rig;
}
elsif ($lef > $rig)
{
$type .= "3'";
$self->{'inside_cut'} = $rig;
$self->{'outside_cut'} = $lef;
}
elsif ($lef == $rig)
{
$type .= "b";
}
$self->{'onebpoverhang'} = 1 if (abs($lef - $rig) == 1);
$self->{'type'} = $type;
$self->{'temp'} = $temp if ($temp);
if ($tempin)
{
my ($intime, $intemp) = split q{@}, $tempin;
$self->{'tempin'} = $intemp;
$self->{'timein'} = $intime;
}
$self->{'score'} = $score if ($score);
$self->{'aggress'} = $aggress;
$self->{'staract'} = 1 if ($staract);
if (exists $methtrans{$methdam})
{
$self->{'methdam'} = $methtrans{$methdam};
}
else
{
$self->{'methdam'} = "indifferent";
}
if (exists $methtrans{$methdcm})
{
$self->{'methdcm'} = $methtrans{$methdcm};
}
else
{
$self->{'methdcm'} = "indifferent";
}
if (exists $methtrans{$methcpg})
{
$self->{'methcpg'} = $methtrans{$methcpg};
}
else
{
$self->{'methcpg'} = "indifferent";
}
if ($vendors)
{
my %vhsh = ();
foreach my $v (split(q{}, $vendors))
{
$vhsh{$v} = $RE_vendors{$v} if (exists $RE_vendors{$v});
carp("$v not in vendor list!") unless (exists $RE_vendors{$v});
}
$self->{'vendors'} = \%vhsh;
}
$self->{'buffers'} = $buffers if ($buffers);
}
$self->{'start'} = $start if ($start);
$self->{'exclude'} = $exclude if ($exclude);
return $self;
}
=head1 FUNCTIONAL METHODS
=head2 clone
By default in GeneDesign code, RestrictionEnzyme objects are meant to stand as
abstracts - that is, they stand for BamHI in general, and not for a particular
instance of a BamHI recognition site. If you want to use the objects in the
latter sense, you will need to clone the abstract object instantiated when the
definition file is read in, thus generating an arbitrary number of BamHI
instances that can then be differentiated by their start attributes.
=cut
sub clone
{
my ($self) = @_;
my $copy;
foreach my $key (keys %$self)
{
if (ref $self->{$key} eq "ARRAY")
{
$copy->{$key} = [@{$self->{$key}}];
}
elsif (ref $self->{$key} eq "HASH")
{
$copy->{$key} = {%{$self->{$key}}};
}
else
{
$copy->{$key} = $self->{$key};
}
}
bless $copy, ref $self;
return $copy;
}
=head2 positions
Generates a hash describing the positions of the enzyme's recognition
sites in a nucleotide sequence. Keys are offset in nucleotides, and values are
the recognition site found at said offset as a string.
=cut
sub positions
{
my ($self, $seq) = @_;
my $total = {};
foreach my $sit (@{$self->{regex}})
{
while ($seq =~ /(?=($sit))/ixg)
{
$total->{pos $seq} = $1;
}
}
return $total;
}
=head2 overhang
Given a nucleotide sequence context, what overhang does this enzyme leave, and
how far away from the cutsite is it?
Arguments:
=cut
sub overhang
{
my ($self, $dna, $context, $strand) = @_;
my ($ohangstart, $mattersbit) = (0, q{});
my $lef;
my $rig;
if ($self->{class} eq "IIP")
{
($lef, $rig) = (length($1), length($2)) if ($self->{cutseq} =~ $IIPreg);
($lef, $rig) = ($rig, $lef) if ($rig < $lef);
$ohangstart = $lef + 1;
$mattersbit = substr($dna, $ohangstart-1, $rig-$lef);
}
elsif ($self->{class} eq "IIA")
{
($lef, $rig) = ($1, $2) if ($self->{cutseq} =~ $IIAreg);
($lef, $rig) = ($rig, $lef) if ($rig < $lef);
if ($strand == 1)
{
$ohangstart = length($dna) + $lef + 1;
}
else
{
$ohangstart = length($context) - length($dna) - $rig + 1;
}
$mattersbit = substr($context, $ohangstart-1, $rig-$lef);
$ohangstart = $strand == 1 ? length($dna) + $lef : 0 - ($rig);
}
else
{
return ();
}
return ($ohangstart, $mattersbit);
}
=head2 display
Generates a tab delimited display string that can be used to print enzyme
information out in a tabular format.
=cut
sub display
{
my ($self) = @_;
my $staract = $self->{staract} ? "*" : q{};
my (@blocked, @inhibed) = ((), ());
push @blocked, "cpg" if ($self->{methcpg} eq "blocked");
push @blocked, "dam" if ($self->{methdam} eq "blocked");
push @blocked, "dcm" if ($self->{methdcm} eq "blocked");
push @inhibed, "cpg" if ($self->{methcpg} eq "inhibited");
push @inhibed, "dam" if ($self->{methdam} eq "inhibited");
push @inhibed, "dcm" if ($self->{methdcm} eq "inhibited");
my $buffstr = undef;
foreach (sort keys %{$self->{buffers}})
{
$buffstr .= "$_ (" . $self->{buffers}->{$_} . ") " if ($self->{buffers}->{$_});
}
my $vendstr = join(", ", values %{$self->{vendors}});
my $display = undef;
my $inact = $self->{tempin} ? " (". $self->{timein} . q{@} . $self->{tempin} . ")" : q{};
$display .= $self->{id} . "\t";
$display .= $self->{cutseq} . $staract . "\t";
$display .= $self->{type} . "\t";
$display .= $self->{start} . "\t" if ($self->{start});
$display .= $self->{temp} . $inact . "\t";
$display .= join(", ", @blocked) . "\t";
$display .= join(", ", @inhibed) . "\t";
$display .= $self->{score} . "\t";
$display .= $buffstr . "\t";
$display .= $vendstr . "\t";
return $display;
}
=head2 common_buffers
Returns an array reference listing the buffers, if any, in which two enzymes
both have 100% activity. in boolean mode returns the number of buffers
=cut
sub common_buffers
{
my ($self, $buddy, $bool) = @_;
$self->throw("Argument is not a Bio::GeneDesign::RestrictionEnzyme")
unless $buddy->isa("Bio::GeneDesign::RestrictionEnzyme");
my $sbuffs = $self->{buffers};
my $bbuffs = $buddy->{buffers};
my @answer;
foreach my $skey (sort keys %{$sbuffs})
{
my $sval = $sbuffs->{$skey};
my $bval = $bbuffs->{$skey};
if ($skey eq "Other" && $sval && $bval && "$sval" eq "$bval")
{
push @answer, $skey;
}
elsif ($sval && $bval && "$sval" == 100 && "$bval" == 100)
{
push @answer, $skey;
}
}
return $bool ? scalar(@answer) : \@answer;
}
=head2 acceptable_buffer
Returns a buffer in which both enzymes will have at least a thresholded amount
of activity.
=cut
sub acceptable_buffer
{
my ($self, $buddy, $level) = @_;
$self->throw("Argument is not a Bio::GeneDesign::RestrictionEnzyme")
unless $buddy->isa("Bio::GeneDesign::RestrictionEnzyme");
$level = $level || 75;
my $sbuffs = $self->{buffers};
my $bbuffs = $buddy->{buffers};
my %answers;
foreach my $skey (sort keys %{$sbuffs})
{
my $sval = $sbuffs->{$skey};
my $bval = $bbuffs->{$skey};
if ($skey eq "Other" && $sval && $bval && $sval == $bval)
{
$answers{$skey} = 200;
}
elsif ($sval && $bval && $sval >= $level && $bval >= $level)
{
$answers{$skey} = $sval + $bval;
}
}
my @keys = sort {$answers{$b} <=> $answers{$a} && $b cmp $a} keys %answers;
return scalar @keys ? $keys[0] : undef;
}
=head2 units
Returns the number of units needed to cleave some sequence
=cut
sub units
{
my ($self, @args) = @_;
my ($buffer, $sequence) = $self->_rearrange([qw(buffer sequence)], @args);
my $poshsh = $self->positions($sequence);
my $count = scalar keys %{$poshsh};
my $freq = $count / (length $sequence);
my $aggr = $self->aggress() || .000001;
$aggr = 1 / $aggr;
$buffer = $buffer || $self->acceptable_buffer($self, 100);
my $buff = $self->buffers->{$buffer} || 1;
my $jad = $buff / 100;
my $adj = $jad > 0 ? 1 / $jad : 0;
my $units = sprintf("%.1f", $freq * $aggr * $adj);
return $units;
}
=head1 FILTERING METHODS
=head2 filter_by_sequence
Arguments: an arrayref of string nucleotide sequences (may be ambiguous)
a flag indicating whether or not the sequences in the array are
required (1 means they must NOT match; default 0 means they must
match)
Returns : 1 if the enzyme passes;
0 if the enzyme fails.
=cut
sub filter_by_sequence
{
my ($self, $arrref, $req) = @_;
$req = 0 if (! $req);
my $result = 1;
foreach my $seq (@$arrref)
{
my $regex = _regres($seq, 1);
if ($regex =~ /\[ X \]/x)
{
print "\tWARNING: Can't parse sequence $seq containing non-nucleotide "
. "characters - ignoring.\n";
next;
}
$result = 0 if ( $req == 1 && $self->{recseq} =~ $regex );
$result = 0 if ( $req == 0 && $self->{recseq} !~ $regex );
}
return $result;
}
=head2 filter_by_score
Arguments : a float
Returns : 1 if the enzyme's score is less than or equal to the argument,
0 if the enzyme's score is higher.
=cut
sub filter_by_score
{
my ($self, $score) = @_;
my $result = 1;
$result = 0 if ($self->{score} > $score);
return $result;
}
=head2 filter_by_vendor
Arguments : an arrayref of vendor abbreviations; see vendor().
Returns : 1 if the enzyme is supplied by any of the vendors queried,
0 else.
=cut
sub filter_by_vendor
{
my ($self, $vendlist) = @_;
my $result = 1;
my $flag = 0;
foreach my $vend (@$vendlist)
{
unless (exists($RE_vendors{$vend}))
{
print "\tWARNING: Can't parse vendor argument $vend - ignoring.\n";
next;
}
$flag++ if ( exists( $self->{vendors}->{$vend} ) );
}
$result = $flag == 0 ? 0 : 1;
return $result;
}
=head2 filter_by_buffer_activity
Arguments : a hashref of buffer thresholds; the key is the buffer name, the
value is an activity threshold.
Returns : 1 if the enzyme meets all the buffer requirements,
0 else.
=cut
sub filter_by_buffer_activity
{
my ($self, $hshref) = @_;
my $result = 1;
my $rebuff = $self->{buffers};
foreach my $buff (keys %$hshref)
{
my $val = $hshref->{$buff};
$result = 0 if ( ! exists($rebuff->{$buff}) || $rebuff->{$buff} < $val );
}
return $result;
}
=head2 filter_by_dcm_sensitivity
Arguments : an arrayref of sensitivity values; the key is the sensitivity
blocked, inhibited, or indifferent
Returns : 1 if the enzyme meets the dcm sensitivity requirements,
0 else.
=cut
sub filter_by_dcm_sensitivity
{
my ($self, $arrref) = @_;
my $result = 1;
my %sensehsh;
foreach my $sense (@$arrref)
{
if ($sense ne "blocked" && $sense ne "inhibited" && $sense ne "indifferent")
{
$sense = lc $sense;
print "\tWARNING: Can't parse dcmsense argument $sense - ignoring.\n";
next;
}
$sensehsh{$sense}++;
}
$result = 0 unless ( exists($sensehsh{$self->{methdcm}}) );
return $result;
}
=head2 filter_by_dam_sensitivity
Arguments : an arrayref of sensitivity values; the key is the sensitivity
blocked, inhibited, or indifferent
Returns : 1 if the enzyme meets the dam sensitivity requirements,
0 else.
=cut
sub filter_by_dam_sensitivity
{
my ($self, $arrref) = @_;
my $result = 1;
my %sensehsh;
foreach my $sense (@$arrref)
{
if ($sense ne "blocked" && $sense ne "inhibited" && $sense ne "indifferent")
{
$sense = lc $sense;
print "\tWARNING: Can't parse damsense argument $sense - ignoring.\n";
next;
}
$sensehsh{$sense}++;
}
$result = 0 unless ( exists($sensehsh{$self->{methdam}}) );
return $result;
}
=head2 filter_by_cpg_sensitivity
Arguments : an arrayref of sensitivity values; the key is the sensitivity
blocked, inhibited, or indifferent
Returns : 1 if the enzyme meets the cpg sensitivity requirements,
0 else.
=cut
sub filter_by_cpg_sensitivity
{
my ($self, $arrref) = @_;
my $result = 1;
my %sensehsh;
foreach my $sense (@$arrref)
{
if ($sense ne "blocked" && $sense ne "inhibited" && $sense ne "indifferent")
{
$sense = lc $sense;
print "\tWARNING: Can't parse cpgsense argument $sense - ignoring.\n";
next;
}
$sensehsh{$sense}++;
}
$result = 0 unless ( exists($sensehsh{$self->{methcpg}}) );
return $result;
}
=head2 filter_by_star_activity
Arguments : 1 if star activity required, 0 else
Returns : 1 if the enzyme meets the star activity requirements,
0 else.
=cut
sub filter_by_star_activity
{
my ($self, $star) = @_;
my $result = 1;
$star = 0 unless ($star);
$result = 0 if (($star && ! $self->{staract}) || (! $star && $self->{staract}));
return $result;
}
=head2 filter_by_incubation_temperature
Arguments : an arrayref of acceptable integer incubation temperatures
Returns : 1 if the enzyme meets the incubation temperature requirements,
0 else.
=cut
sub filter_by_incubation_temperature
{
my ($self, $arrref) = @_;
my $result = 1;
my %temps;
foreach my $temp (@$arrref)
{
if ($temp !~ /\d/x || $temp <= 0)
{
print "\tWARNING: Can't parse incubation argument $temp - ignoring.\n";
}
$temps{$temp}++;
}
$result = 0 unless ( exists $temps{$self->{temp}});
return $result;
}
=head2 filter_by_inactivation_temperature
Arguments : an acceptable integer inactivation temperature maximum
Returns : 1 if the enzyme meets the inactivation temperature requirement,
0 else.
=cut
sub filter_by_inactivation_temperature
{
my ($self, $temp) = @_;
my $result = 1;
if ($temp !~ /\d/x || $temp <= 0)
{
print "\tWARNING: Can't parse inactivation argument $temp - ignoring.\n";
}
else
{
$result = 0 if ($self->{tempin} > $temp);
}
return $result;
}
=head2 filter_by_base_ambiguity
Arguments : "nonNonly" if any non N bases are allowed; "ATCGonly" if only
A, T, C, or G are allowed
Returns : 1 if the enzyme meets the ambiguous nucleotide requirement,
0 else.
=cut
sub filter_by_base_ambiguity
{
my ($self, $ambig) = @_;
my $result = 1;
if ($ambig ne "nonNonly" && $ambig ne "ATCGonly")
{
print "\tWARNING: Can't parse ambiguity argument $ambig - ignoring.\n";
}
else
{
my $ambregex;
$ambregex = qr/N/ if ($ambig eq "nonNonly");
$ambregex = $ambnt if ($ambig eq "ATCGonly");
$result = 0 unless ( $self->{recseq} =~ $ambregex );
}
return $result;
}
=head2 filter_by_length
Arguments : an arrayref of acceptable recognition site lengths
Returns : 1 if the enzyme meets the recognition site length requirements,
0 else.
=cut
sub filter_by_length
{
my ($self, $arrref) = @_;
my $result = 1;
my %lens;
foreach my $len (@$arrref)
{
if ($len =~ /\D/x || $len <= 0)
{
print "\tWARNING: Can't parse length argument $len - ignoring.\n";
next;
}
$lens{$len}++;
}
$result = 0 unless ( exists $lens{length($self->{recseq})} );
return $result;
}
=head2 filter_by_overhang_palindromy
Arguments : an arrayref of acceptable overhang palindromys, from the list
pal (palindromic),
nonpal (nonpalindromic),
pnon (potentially nonpalindromic)
Returns : 1 if the enzyme meets the palindromy requirements,
0 else.
=cut
sub filter_by_overhang_palindromy
{
my ($self, $arrref) = @_;
my $result = 1;
my %pals;
foreach my $pal (@$arrref)
{
if ($pal ne "pal" && $pal ne "pnon" && $pal ne "nonpal")
{
print "\tWARNING: Can't parse palindromy argument $pal - ignoring.\n";
next;
}
$pals{$pal}++;
}
$result = 0 unless (exists $pals{$self->{palindromy}});
return $result;
}
=head2 filter_by_stickiness
Arguments : an arrayref of acceptable overhang orientations, from the list
1 (single basepair overhang),
5 (five prime overhang),
3 (three prime overhang),
b (blunt ended)
Returns : 1 if the enzyme meets the overhang requirements,
0 else.
=cut
sub filter_by_stickiness
{
my ($self, $arrref) = @_;
my $result = 1;
my %sticks;
foreach my $stick (@$arrref)
{
if ($stick ne "5" && $stick ne "3" && $stick ne "1" && $stick ne "b")
{
print "\tWARNING: Can't parse sticky argument $stick - ignoring.\n";
next;
}
$sticks{$stick}++;
}
$result = 0 if ($self->{onebpoverhang} && ! exists $sticks{1});
my $type = $self->{type};
$type =~ s/\'//xg;
$result = 0 unless (exists $sticks{$type});
return $result;
}
=head1 ACCESSOR METHODS
Methods for setting and accessing enzyme attributes
=head2 id
The name of the enzyme.
=cut
sub id
{
my ($self) = @_;
return $self->{'id'};
}
=head2 display_name
The name of the enzyme.
=cut
sub display_name
{
my ($self) = @_;
return $self->{'id'};
}
=head2 score
This attribute initially holds the price in dollars per unit of the enzyme
(2009 US Dollars) but can be used to hold any score or cost value.
=cut
sub score
{
my ($self, $value) = @_;
if (defined $value)
{
$self->{'score'} = $value;
}
return $self->{'score'};
}
=head2 aggress
Aggressiveness is the number of recognition sites in a template piece of DNA
(usually lambda, but sometimes adeno2, pBR322, pUC19, pXba, etc) over the total
length of that template piece of DNA. This number tells the manufacturer how
much enzyme to sell as a "unit" - the amount of enzyme required to fully digest
one microgram of template DNA under reaction conditions in an hour.
=cut
sub aggress
{
my ($self, $value) = @_;
if (defined $value)
{
$self->{'aggress'} = $value;
}
return $self->{'aggress'};
}
=head2 len
The length in bases of the recognition sequence (recseq).
=cut
sub len
{
my ($self) = @_;
return $self->{'length'};
}
=head2 methcpg
The effect of CpG methylation on the enzyme's efficacy.
=cut
sub methcpg
{
my ($self) = @_;
return $self->{'methcpg'};
}
=head2 methdcm
The effect of Dcm methylation on the enzyme's efficacy.
=cut
sub methdcm
{
my ($self) = @_;
return $self->{'methdcm'};
}
=head2 methdam
The effect of Dam methylation on the enzyme's efficacy.
=cut
sub methdam
{
my ($self) = @_;
return $self->{'methdam'};
}
=head2 buffers
A hash reference where the keys are buffer names and the values are the activity
level of the enzyme in that Buffer. Since most of the enzymes in the default
GeneDesign list are NEB enzymes, this is usually full of NEB buffers.
=cut
sub buffers
{
my ($self) = @_;
return $self->{'buffers'};
}
=head2 vendors
A hash reference where the keys are abbreviations for and the values are names
of vendors that stock the enzyme. These are read in from the enzyme file.
B = Invitrogen
C = Minotech
E = Stratagene
F = Thermo Scientific Fermentas
I = SibEnzyme
J = Nippon Gene Co.
K = Takara
M = Roche Applied Science
N = New England Biolabs
O = Toyobo Technologies
Q = Molecular Biology Resources
R = Promega
S = Sigma Aldrich
U = Bangalore Genei
V = Vivantis
X = EURx
Y = CinnaGen
=cut
sub vendors
{
my ($self) = @_;
return $self->{'vendors'};
}
=head2 tempin
The temperature in degrees Celsius that deactivates the enzyme.
=cut
sub tempin
{
my ($self) = @_;
return $self->{'tempin'};
}
=head2 timein
The time required at inactivation temperature to deactivate the enzyme.
=cut
sub timein
{
my ($self) = @_;
return $self->{'timein'};
}
=head2 temp
Incubation temperature for the best enzyme activity, in degrees Celsius.
=cut
sub temp
{
my ($self) = @_;
return $self->{'temp'};
}
=head2 recseq
This attribute is the "clean" description of the enzyme's recognition sequence -
that is, no information about cleavage site can be gained from this attribute.
This is determined automatically from the cleavage string (cutseq) at
instantiation.
=cut
sub recseq
{
my ($self) = @_;
return $self->{'recseq'};
}
=head2 seq
Synonym for recseq
=cut
sub seq
{
my ($self) = @_;
return $self->{'recseq'};
}
=head2 cutseq
This attribute is the string description of the enzyme's recognition sequence.
It includes information about both the recognition and cleavage sites.
See http://rebase.neb.com/rebase/rebrec.html for help interpreting this field.
=cut
sub cutseq
{
my ($self) = @_;
return $self->{'cutseq'};
}
=head2 regex
This attribute stores a set of regular expressions as an array reference to
speed the search for recognition sites in sequence. The first entry in the
arrayref is the regular expression representing the forward orientation of
the recognition sequence; the second entry represents the reverse orientation
and is only defined if the recognition site is nonpalindromic.
This attribute is defined at instantiation.
=cut
sub regex
{
my ($self) = @_;
return $self->{'regex'};
}
=head2 class
Class describes the cutting behavior of an enzyme. The classes used by
GeneDesign uses a generalized subset of the classes as described at Rebase - for
the purposes of enzyme editing, three classes have so far proven to be enough.
See http://rebase.neb.com/cgi-bin/sublist for the full description of enzyme
classes.
IIP : This enzyme has a symmetric target and a symmetric cleavage site; this
usually means that the enzyme cleaves inside its own recognition site.
This is not the same as overhang palindromy!
IIA : This enzyme has an asymmetric recognition site and usually cleaves
outside of it.
IIB : This enzyme has one recognition site and two cleavage sites, one on
either side of the recognition site, and thus cuts itself out of
sequence.
=cut
sub class
{
my ($self) = @_;
return $self->{'class'};
}
=head2 classex
=cut
sub classex
{
my ($self) = @_;
return $self->{'classex'};
}
=head2 class_regexes
Short cut to accessing class regular expressions
=cut
sub class_regexes
{
return {"IIP" => $IIPreg, "IIA" => $IIAreg, "IIB" => $IIBreg};
}
=head2 type
Type describes the kind of overhang left by an enzyme. This is probably not a
good use of the word type.
Type may be 5', for a five prime overhang; 3', for a three prime overhang;
or b for blunt ends.
=cut
sub type
{
my ($self) = @_;
return $self->{'type'};
}
=head2 onebpoverhang
One basepair overhangs can be harder to ligate than blunt ends. This attribute
returns 1 if an enzyme leaves a 1bp overhang and 0 else.
=cut
sub onebpoverhang
{
my ($self) = @_;
return $self->{'onebpoverhang'};
}
=head2 exclude
Some enzymes share overlapping recognition sites. If you are trying to ensure
the absence or uniqueness of a recognition site, you will want to be sure to
exclude isoschizomers and neoschizomers from consideration elsewhere. The
exclude attribute stores an array reference that lists the ids of neo- and
isoschizomers - or any arbitrary enzyme that is incompatible with this enzyme -
for easy lookup.
=cut
sub exclude
{
my ($self, $value) = @_;
if (defined $value)
{
$self->throw("$value is not a reference to an array")
unless (ref $value eq "ARRAY");
$self->{'exclude'} = $value;
}
return $self->{'exclude'};
}
=head2 palindromy
Information about the overhang the enzyme leaves.
pal = palindromic
nonpal = nonpalindromic
pnon = potentially nonpalindromic, or sometimes palindromic and sometimes
nonpalindromic
unknown = unknown
=cut
sub palindromy
{
my ($self) = @_;
return $self->{'palindromy'};
}
=head2 staract
1 if the enzyme exhibits star activity, 0 else
=cut
sub staract
{
my ($self) = @_;
return $self->{'staract'};
}
=head2 start
The offset in nucleotides of the enzymes recognition site in an ORF
=cut
sub start
{
my ($self, $value) = @_;
if (defined $value)
{
$self->{'start'} = $value;
}
return $self->{'start'};
}
=head2 outside_cut
=cut
sub outside_cut
{
my ($self) = @_;
return $self->{'outside_cut'};
}
=head2 inside_cut
=cut
sub inside_cut
{
my ($self) = @_;
return $self->{'inside_cut'};
}
1;
__END__
=head1 COPYRIGHT AND LICENSE
Copyright (c) 2013, GeneDesign developers
All rights reserved.
Redistribution and use in source and binary forms, with or without modification,
are permitted provided that the following conditions are met:
* Redistributions of source code must retain the above copyright notice, this
list of conditions and the following disclaimer.
* Redistributions in binary form must reproduce the above copyright notice, this
list of conditions and the following disclaimer in the documentation and/or
other materials provided with the distribution.
* The names of Johns Hopkins, the Joint Genome Institute, the Lawrence Berkeley
National Laboratory, the Department of Energy, and the GeneDesign developers may
not be used to endorse or promote products derived from this software without
specific prior written permission.
THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS "AS IS" AND
ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED
WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE
DISCLAIMED. IN NO EVENT SHALL THE DEVELOPERS BE LIABLE FOR ANY DIRECT, INDIRECT,
INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT
LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR
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LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE
OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF
ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.
=cut