Bio::Tools
RestrictionEnzyme
Summary
Bio::Tools::RestrictionEnzyme - Bioperl object for a restriction endonuclease (cuts DNA at specific locations)
Package variables
Privates (from "my" definitions)
%RE = ( 'AatII' => 'GACGTC 5', 'AccI' => 'GTMKAC 2', 'AclI' => 'AACGTT 2', 'AcyI' => 'GRCGYC 2', 'AflII' => 'CTTAAG 1', 'AflIII' => 'ACRYGT 1', 'AgeI' => 'ACCGGT 1', 'AhaIII' => 'TTTAAA 3', 'AhdI' => 'GACNNNNNGTC 6', 'AluI' => 'AGCT 2', 'AlwNI' => 'CAGNNNCTG 6', 'ApaBI' => 'GCANNNNNTGC 8', 'ApaI' => 'GGGCCC 5', 'ApaLI' => 'GTGCAC 1', 'ApoI' => 'RAATTY 1', 'AscI' => 'GGCGCGCC 2', 'AsuI' => 'GGNCC 1', 'AsuII' => 'TTCGAA 2', 'AvaI' => 'CYCGRG 1', 'AvaII' => 'GGWCC 1', 'AvrII' => 'CCTAGG 1', 'BalI' => 'TGGCCA 3', 'BamHI' => 'GGATCC 1', 'BclI' => 'TGATCA 1', 'BetI' => 'WCCGGW 1', 'BglI' => 'GCCNNNNNGGC 7', 'BglII' => 'AGATCT 1', 'BsaAI' => 'YACGTR 3', 'BsaBI' => 'GATNNNNATC 5', 'BsePI' => 'GCGCGC 1', 'BsiYI' => 'CCNNNNNNNGG 7', 'Bsp1407I'=> 'TGTACA 1', 'BspHI' => 'TCATGA 1', 'BspLU11I'=> 'ACATGT 1', 'BspMII' => 'TCCGGA 1', 'BstEII' => 'GGTNACC 1', 'BstXI' => 'CCANNNNNNTGG 8', 'Cac8I' => 'GCNNGC 3', 'CauII' => 'CCSGG 2', 'Cfr10I' => 'RCCGGY 1', 'CfrI' => 'YGGCCR 1', 'ClaI' => 'ATCGAT 2', 'CviJI' => 'RGCY 2', 'CviRI' => 'TGCA 2', 'DdeI' => 'CTNAG 1', 'DpnI' => 'GATC 2', 'DraI' => 'TTTAAA 3', 'DraII' => 'RGGNCCY 2', 'DraIII' => 'CACNNNGTG 6', 'DrdI' => 'GACNNNNNNGTC 7', 'DsaI' => 'CCRYGG 1', 'Eam1105I'=> 'GACNNNNNGTC 6', 'Eco47III'=> 'AGCGCT 3', 'EcoNI' => 'CCTNNNNNAGG 5', 'EcoRI' => 'GAATTC 1', 'EcoRII' => 'CCWGG 0', 'EcoRV' => 'GATATC 3', 'EspI' => 'GCTNAGC 2', 'Fnu4HI' => 'GCNGC 2', 'FnuDII' => 'CGCG 2', 'FseI' => 'GGCCGGCC 6', 'HaeI' => 'WGGCCW 3', 'HaeII' => 'RGCGCY 5', 'HaeIII' => 'GGCC 2', 'HgiAI' => 'GWGCWC 5', 'HgiCI' => 'GGYRCC 1', 'HgiJII' => 'GRGCYC 5', 'HhaI' => 'GCGC 3', 'HincII' => 'GTYRAC 3', 'HindII' => 'GTYRAC 3', 'HindIII' => 'AAGCTT 1', 'HinfI' => 'GANTC 1', 'HpaI' => 'GTTAAC 3', 'HpaII' => 'CCGG 1', 'KpnI' => 'GGTACC 5', 'MaeI' => 'CTAG 1', 'MaeII' => 'ACGT 1', 'MaeIII' => 'GTNAC 0', 'MboI' => 'GATC 0', 'McrI' => 'CGRYCG 4', 'MfeI' => 'CAATTG 1', 'MluI' => 'ACGCGT 1', 'MseI' => 'TTAA 1', 'MslI' => 'CAYNNNNRTG 5', 'MstI' => 'TGCGCA 3', 'MwoI' => 'GCNNNNNNNGC 7', 'NaeI' => 'GCCGGC 3', 'NarI' => 'GGCGCC 2', 'NcoI' => 'CCATGG 1', 'NdeI' => 'CATATG 2', 'NheI' => 'GCTAGC 1', 'NlaIII' => 'CATG 4', 'NlaIV' => 'GGNNCC 3', 'NotI' => 'GCGGCCGC 2', 'NruI' => 'TCGCGA 3', 'NspBII' => 'CMGCKG 3', 'NspI' => 'RCATGY 5', 'PacI' => 'TTAATTAA 5', 'PflMI' => 'CCANNNNNTGG 7', 'PmaCI' => 'CACGTG 3', 'PmeI' => 'GTTTAAAC 4', 'PpuMI' => 'RGGWCCY 2', 'PshAI' => 'GACNNNNGTC 5', 'PstI' => 'CTGCAG 5', 'PvuI' => 'CGATCG 4', 'PvuII' => 'CAGCTG 3', 'RsaI' => 'GTAC 2', 'RsrII' => 'CGGWCCG 2', 'SacI' => 'GAGCTC 5', 'SacII' => 'CCGCGG 4', 'Sau96I' => 'GGNCC 1', 'SalI' => 'GTCGAC 1', 'SanDI' => 'GGGWCCC 2', 'SauI' => 'CCTNAGG 2', 'SbfI' => 'CCTGCAGG 6', 'ScaI' => 'AGTACT 3', 'ScrFI' => 'CCNGG 2', 'SduI' => 'GDGCHC 5', 'SecI' => 'CCNNGG 1', 'SexAI' => 'ACCWGGT 1', 'SfeI' => 'CTRYAG 1', 'SfiI' => 'GGCCNNNNNGGCC 8', 'SgfI' => 'GCGATCGC 5', 'SgrAI' => 'CRCCGGYG 2', 'SmaI' => 'CCCGGG 3', 'SmlI' => 'CTYRAG 1', 'SnaBI' => 'TACGTA 3', 'SpeI' => 'ACTAGT 1', 'SphI' => 'GCATGC 5', 'SplI' => 'CGTACG 1', 'SrfI' => 'GCCCGGGC 4', 'Sse8387I'=> 'CCTGCAGG 6', 'Sse8647I'=> 'AGGWCCT 2', 'SspI' => 'AATATT 3', 'StuI' => 'AGGCCT 3', 'StyI' => 'CCWWGG 1', 'SwaI' => 'ATTTAAAT 4', 'TaqI' => 'TCGA 1', 'TatI' => 'WGTACW 1', 'TfiI' => 'GAWTC 1', 'TseI' => 'GCWGC 1', 'Tsp45I' => 'GTSAC 0', 'Tsp4CI' => 'ACNGT 3', 'TspEI' => 'AATT 0', 'TspRI' => 'CASTGNN 7', 'Tth111I' => 'GACNNNGTC 4', 'VspI' => 'ATTAAT 2', 'XbaI' => 'TCTAGA 1', 'XcmI' => 'CCANNNNNNNNNTGG 8', 'XhoI' => 'CTCGAG 1', 'XhoII' => 'RGATCY 1', 'XmaIII' => 'CGGCCG 1', 'XmnI' => 'GAANNNNTTC 5', )
Included modules
Inherit
Synopsis
require Bio::Tools::RestrictionEnzyme;
## Create a new object by name.
$re1 = new Bio::Tools::RestrictionEnzyme(-NAME =>'EcoRI');
## Create a new object using special syntax
## which specifies the enzyme name, recognition site, and cut position.
## Used for enzymes not known to this module.
$re2 = new Bio::Tools::RestrictionEnzyme(-NAME =>'EcoRV--GAT^ATC',
-MAKE =>'custom');
## Get a list of names of all available restriction enzymes
## known to this module.
@all = $re->available_list();
## Get the names of restriction enzymes that have 6 bp
## recognition sequences
@sixcutters = $re->available_list(6);
Description
The Bio::Tools::RestrictionEnzyme.pm module encapsulates generic data and
methods for using restriction endonucleases for in silico restriction
analysis of DNA sequences.
This module is a precursor for a more full featured version that may do such things as
download data from online databases such as REBase
http://www.neb.com/rebase/.
Thus, there is currently no functionality for obtaining data about commercial
availability for a restriction enzyme.
At some point in the future, it may make sense to derive RestrictionEnzymes from
a class such as Bio::Enzyme or Bio::Prot::Protein (neither of which now exist)
so that more data about the enzyme and related information can be easily obtained.
This module is currently in use at
http://genome-www.stanford.edu/Sacch3D/analysis/
To digest a sequence on runs of N's in the sequence. Here's what you can do:
$re_n = new Bio::Tools::RestrictionEnzyme(-name=>'N--NNNNN', -make=>'custom');
Specify the number of N's you want to match in the -name parameter.
So the above example will recognize and cut at runs of 5 Ns.
If you wanted to cut at runs of 10 N's, you would use
-name => 'N--NNNNNNNNNN'
Note that you must use a specific number of N's, you cannot use a regexp to digest at N+ for example,
because the actual number of N's at each site are not recorded when the sequence is analyzed.
So cut_locations( ) wouldn't be correct.
Methods
Methods description
Title : new
Purpose : Initializes the RestrictionEnzyme object and calls
: superclass constructor last (Bio:Seq.pm).
Returns : n/a
Argument : Parameters passed to new()
Comments : A RestrictionEnzyme object manages its recognition sequence
: as a Bio::PrimarySeq object.
See Also : _make_custom(), _make_standard(), Bio::PrimarySeq.pm::_initialize() |
Title : cuts_after
Usage : $num = $re->cuts_after();
Purpose : Sets/Gets an integer indicating the position of cleavage
: relative to the 5' end of the recognition sequence.
Returns : Integer
Argument : Integer (optional)
Throws : Exception if argument is non-numeric.
Access : Public
Comments : This method is only needed to change the cuts at
: position. This data is automatically set during
: construction.
See Also : _make_standard(), _make_custom() |
Title : site
Usage : $re->site();
Purpose : Gets the recognition sequence for the enzyme.
Example : $seq_string = $re->site();
Returns : String containing recognition sequence indicating
: cleavage site as in 'G^AATTC'.
Argument : n/a
Throws : n/a
Comments : If you want a simple string representing the site without
any '^', use the string() method.
See Also : string() |
Title : seq
Usage : $re->seq();
Purpose : Get the Bio::PrimarySeq.pm-derived object representing
: the recognition sequence
Returns : String
Argument : n/a
Throws : n/a
See Also : string(), revcom() |
Title : string
Usage : $re->string();
Purpose : Get a string representing the recognition sequence.
Returns : String. Does NOT contain a '^' representing the cut location
as returned by the site() method
Argument : n/a
Throws : n/a
Comments : Delegates to the Bio::PrimarySeq-derived object.
See Also : seq(), site(), revcom() |
Title : revcom
Usage : $re->revcom();
Purpose : Get a string representing the reverse complement of
: the recognition sequence.
Returns : String
Argument : n/a
Throws : n/a
Comments : Delegates to the Bio::PrimarySeq.pm-derived object, but needs to get
out the string from it, as now Bio::PrimarySeq->revcom makes a Bio::PrimarySeq
object
See Also : seq(), string() |
Title : cut_seq
Usage : $re->cut_seq();
Purpose : Conceptually cut or "digest" a DNA sequence with the given enzyme.
Example : $string = $re->cut_seq();
Returns : List of strings containing the resulting fragments.
Argument : Reference to a Bio::PrimarySeq.pm-derived object.
Throws : Exception if argument is not an object.
: (Does not yet verify that it is derived from Bio::PrimarySeq.pm.)
Comments : Strategy relies on Perl's built-in split() function.
: Since split removes the recognition pattern, the resulting
: fragments must be repaired after split()-ing.
: There is currently no support for partial digestions. |
Title : cut_locations
Usage : my $locations = $re->cut_locations();
Purpose : Report the location of the recognition site(s) within
: an input sequence.
Example : my $locations = $re->annotate_seq($seqObj);
Returns : Arrayref of starting locations where enzyme would cut
Argument : Reference to a Bio::PrimarySeqI-derived sequence object.
Throws : n/a
Comments : |
Title : annotate_seq
Usage : $re->annotate_seq();
Purpose : Identify the location of the recognition site(s) within
: an input sequence. Uses HTML.
Example : $annot_seq = $re->annotate_seq($seqObj);
Returns : String containing the annotated sequence.
Argument : Reference to a Bio::PrimarySeq.pm-derived sequence object.
Throws : n/a
Comments : The annotated sequence must be viewed with a web
: browser to see the location(s) of the recognition site(s). |
Title : palindromic
Usage : $re->palindromic();
Purpose : Determines if the recognition sequence is palindromic
: for the current restriction enzyme.
Returns : Boolean
Argument : n/a
Throws : n/a
Access : Public
Comments : A palindromic site (EcoRI): 5-GAATTC-3
: 3-CTTAAG-5 |
Title : is_available
Usage : $re->is_available();
Purpose : Determine if an enzyme is available (to this module).
: (see the package lexical %RE).
Example : $re->is_available('EcoRI');
: &Bio::Tools::RestrictionEnzyme::is_available($object,'EcoRI');
Returns : Boolean
Argument : String
Throws : n/a
Comments : This method does NOT give information about
: commercial availability (yet).
: Enzyme names are CASE SENSITIVE.
See Also : available_list() |
Title : name
Usage : $obj->name($newval)
Function:
Example :
Returns : value of name
Args : newvalue (optional) |
Title : available_list
Usage : $re->available_list([]);
Purpose : Retrieve a list of currently available enzymes.
Example : @all = $re->available_list(); ## All enzymes
: @six_cutters = $re->available_list(6); ## All 6-cutters
Returns : List of strings
Argument : Integer (optional)
Throws : n/a
Comments : This method may be more appropriate for a REData.pm class.
See Also : is_available() |
Methods code
sub new
{ my($class, @args) = @_;
my $self = $class->SUPER::new(@args);
my ($name,$make) = $self->_rearrange([qw(NAME MAKE)],@args);
$name && $self->name($name);
my %data;
if(defined $make && $make eq 'custom') {
%data = $self->_make_custom($name);
} else {
%data = $self->_make_standard($name);
}
$self->{'_seq'} = new Bio::PrimarySeq(%data,
-VERBOSE =>$self->verbose,
-alphabet => 'dna',
);
return $self; } |
sub _make_standard
{ my($self, $name) = @_;
$name =~ s/^\s+|\s+$//g;
$self->is_available($name) ||
$self->throw("Unavailable or undefined enzyme: $name (Note: CASE SENSITIVE)\n" .
"Currently available enzymes:\n @RE_available\n");
my @data = split( ' ', $RE{$name});
my (%dat);
$dat{-SEQ} = $data[0];
$dat{-NAME} = $dat{-ID}= $name;
$self->{'_cuts_after'} = $data[1];
return %dat; } |
sub _make_custom
{ my($self, $name) = @_;
$name =~ s/\s+//g;
my @parts = split '--', $name;
my (%dat);
$dat{-NAME} = $dat{-ID} = $parts[0];
$self->name($parts[0]);
$parts[1] || return $self->throw("Undefined recognition site for $parts[0].",
"Use this syntax: EcoRV--GAT^ATC");
my $cut_index = index $parts[1], '^';
if( $cut_index <0) { $cut_index = 0;
$self->warn("Unknown cut position for $parts[0]. Assuming position 0\n" .
"Use carat to specify cut position (e.g., G^AATTC)"); }
$self->{'_cuts_after'} = $cut_index;
$parts[1] =~ s/\^//g;
$dat{-SEQ} = $parts[1];
return %dat; } |
sub cuts_after
{ my $self = shift;
if(@_) { my $num = shift;
if($num == 0 and $num ne '0') {
$self->throw("The cuts_after position be an integer ($num)");
}
$self->{'_cuts_after'} = $num;
}
$self->{'_cuts_after'}; } |
sub site
{ my $self = shift;
my $seq = $self->seq;
my $cuts_after = $self->cuts_after;
if($cuts_after > 0) {
return $seq->subseq(1, $self->cuts_after).'^'.$seq->subseq($self->cuts_after+1, $seq->length);
} else {
return $seq->seq;
} } |
sub seq
{ my $self = shift; $self->{'_seq'};} |
sub string
{ my $self = shift; $self->{'_seq'}->seq;} |
sub revcom
{ my $self = shift; $self->{'_seq'}->revcom->seq();} |
sub cut_seq
{ my( $self, $seqObj) = @_;
if( !ref($seqObj) ||
! $seqObj->isa('Bio::PrimarySeqI') ) {
$self->throw( "Can't cut sequence. Missing or invalid object".
"seqObj: $seqObj");
}
my $cuts_after = $self->{'_cuts_after'};
my ($site_3prime_seq, $site_5prime_seq);
my $reSeq = $self->seq;
if($cuts_after == 0) {
$site_3prime_seq = '';
$site_5prime_seq = $reSeq->seq();
} elsif($cuts_after == $reSeq->length) {
$site_3prime_seq = $reSeq->seq();
$site_5prime_seq = '';
} else {
$site_3prime_seq = $reSeq->subseq(1, $self->{'_cuts_after'});
$site_5prime_seq = $reSeq->subseq($self->{'_cuts_after'}+1, $reSeq->length);
}
$self->debug("3' site: $site_3prime_seq\n5' site: $site_5prime_seq\n");
my(@re_frags);
my $seq = uc $self->_expanded_string;
if(!$self->palindromic and $self->name ne 'N') {
my $revseq = $self->_expanded_string( $reSeq->revcom );
$seq .= '|'.uc($revseq);
}
$self->debug(sprintf("$ID: site seq: %s\n\n", $seq));
$self->debug(sprintf("$ID: splitting %s\n\n",$reSeq->seq));
@re_frags = split(/$seq/, uc $seqObj->seq);
$self->debug("$ID: cut_seq, ".scalar @re_frags. " fragments.\n");
my($i);
my $numFrags = scalar @re_frags;
for($i=0; $i<$numFrags; $i++) {
$i < $#re_frags and $re_frags[$i] = $re_frags[$i].$site_3prime_seq;
$i > 0 and $re_frags[$i] = $site_5prime_seq.$re_frags[$i];
}
@re_frags; } |
sub cut_locations
{ my($self, $seqobj) = @_;
my $site = $self->_expanded_string;
my $seq = $seqobj->seq;
study($seq);
my @locations;
while( $seq =~ /($site)/g ) {
push @locations, length($`);
}
return\@ locations; } |
sub _expanded_string
{ my ($self, $str) = @_;
$str ||= $self->string;
if( $self->name ne 'N' ) {
$str =~ s/N|X/\./g;
$str =~ s/R/\[AG\]/g;
$str =~ s/Y/\[CT\]/g;
$str =~ s/S/\[GC\]/g;
$str =~ s/W/\[AT\]/g;
$str =~ s/M/\[AC\]/g;
$str =~ s/K/\[TG\]/g;
$str =~ s/B/\[CGT\]/g;
$str =~ s/D/\[AGT\]/g;
$str =~ s/H/\[ACT\]/g;
$str =~ s/V/\[ACG\]/g;
}
return $str;} |
sub annotate_seq
{ my($self, $seqObj) = @_;
my $site = $self->_expanded_string;
my $seq = $seqObj->seq;
$seq =~ s|$site|<b>$site</b>|g;
return $seq; } |
sub palindromic
{ my $self = shift;
$self->string eq $self->revcom; } |
sub is_available
{ my($self,$name) = @_;
exists $RE{$name}; } |
sub available
{ my($self,$name) = @_;
print STDERR "\nDeprecated method: $ID:: available(); ".
"use is_available() instead.\n";
$self->is_available($name); } |
sub name
{ my ($obj,$value) = @_;
if( defined $value) {
$obj->{'name'} = $value;
}
return $obj->{'name'};} |
sub available_list
{ my($self,$size) = @_;
$size ||= 'all';
$size eq 'all' and return @RE_available;
my(@data, @names);
foreach (@RE_available) {
@data = split /\s/, $RE{$_};
if(length $data[0] == $size) {
push @names, $_;
}
}
@names; } |
General documentation
See the script examples/restriction.pl in the Bioperl distribution.
User feedback is an integral part of the evolution of this and other Bioperl modules.
Send your comments and suggestions preferably to one of the Bioperl mailing lists.
Your participation is much appreciated.
bioperl-l@bioperl.org - General discussion
http://bioperl.org/MailList.shtml - About the mailing lists
Report bugs to the Bioperl bug tracking system to help us keep track the bugs and
their resolution. Bug reports can be submitted via email or the web:
bioperl-bugs@bio.perl.org
http://bio.perl.org/bioperl-bugs/
Steve Chervitz, <sac@bioperl.org>
Copyright (c) 1997-2002 Steve A. Chervitz. All Rights Reserved.
This module is free software; you can redistribute it and/or
modify it under the same terms as Perl itself.
Bio::Root::Root - Base class.
Bio::PrimarySeq - Lightweight sequence object.
http://bio.perl.org/ - Bioperl Project Homepage
Methods beginning with a leading underscore are considered private
and are intended for internal use by this module. They are
not considered part of the public interface and are described here
for documentation purposes only.