Provided by: libbio-perl-perl_1.7.2-2_all 
NAME
Bio::Restriction::Enzyme - A single restriction endonuclease (cuts DNA at specific
locations)
SYNOPSIS
# set up a single restriction enzyme. This contains lots of
# information about the enzyme that is generally parsed from a
# rebase file and can then be read back
use Bio::Restriction::Enzyme;
# define a new enzyme with the cut sequence
my $re=Bio::Restriction::Enzyme->new
(-enzyme=>'EcoRI', -seq=>'G^AATTC');
# once the sequence has been defined a bunch of stuff is calculated
# for you:
#### PRECALCULATED
# find where the enzyme cuts after ...
my $ca=$re->cut;
# ... and where it cuts on the opposite strand
my $oca = $re->complementary_cut;
# get the cut sequence string back.
# Note that site will return the sequence with a caret
my $with_caret=$re->site; #returns 'G^AATTC';
# but it is also a Bio::PrimarySeq object ....
my $without_caret=$re->seq; # returns 'GAATTC';
# ... and so does string
$without_caret=$re->string; #returns 'GAATTC';
# what is the reverse complement of the cut site
my $rc=$re->revcom; # returns 'GAATTC';
# now the recognition length. There are two types:
# recognition_length() is the length of the sequence
# cutter() estimate of cut frequency
my $recog_length = $re->recognition_length; # returns 6
# also returns 6 in this case but would return
# 4 for GANNTC and 5 for RGATCY (BstX2I)!
$recog_length=$re->cutter;
# is the sequence a palindrome - the same forwards and backwards
my $pal= $re->palindromic; # this is a boolean
# is the sequence blunt (i.e. no overhang - the forward and reverse
# cuts are the same)
print "blunt\n" if $re->overhang eq 'blunt';
# Overhang can have three values: "5'", "3'", "blunt", and undef
# Direction is very important if you use Klenow!
my $oh=$re->overhang;
# what is the overhang sequence
my $ohseq=$re->overhang_seq; # will return 'AATT';
# is the sequence ambiguous - does it contain non-GATC bases?
my $ambig=$re->is_ambiguous; # this is boolean
print "Stuff about the enzyme\nCuts after: $ca\n",
"Complementary cut: $oca\nSite:\n\t$with_caret or\n",
"\t$without_caret\n";
print "Reverse of the sequence: $rc\nRecognition length: $recog_length\n",
"Is it palindromic? $pal\n";
print "The overhang is $oh with sequence $ohseq\n",
"And is it ambiguous? $ambig\n\n";
### THINGS YOU CAN SET, and get from rich REBASE file
# get or set the isoschizomers (enzymes that recognize the same
# site)
$re->isoschizomers('PvuII', 'SmaI'); # not really true :)
print "Isoschizomers are ", join " ", $re->isoschizomers, "\n";
# get or set the methylation sites
$re->methylation_sites(2); # not really true :)
print "Methylated at ", join " ", keys %{$re->methylation_sites},"\n";
#Get or set the source microbe
$re->microbe('E. coli');
print "It came from ", $re->microbe, "\n";
# get or set the person who isolated it
$re->source("Rob"); # not really true :)
print $re->source, " sent it to us\n";
# get or set whether it is commercially available and the company
# that it can be bought at
$re->vendors('NEB'); # my favorite
print "Is it commercially available :";
print $re->vendors ? "Yes" : "No";
print " and it can be got from ", join " ",
$re->vendors, "\n";
# get or set a reference for this
$re->reference('Edwards et al. J. Bacteriology');
print "It was not published in ", $re->reference, "\n";
# get or set the enzyme name
$re->name('BamHI');
print "The name of EcoRI is not really ", $re->name, "\n";
DESCRIPTION
This module defines a single restriction endonuclease. You can use it to make custom
restriction enzymes, and it is used by Bio::Restriction::IO to define enzymes in the New
England Biolabs REBASE collection.
Use Bio::Restriction::Analysis to figure out which enzymes are available and where they
cut your sequence.
RESTRICTION MODIFICATION SYSTEMS
At least three geneticaly and biochamically distinct restriction modification systems
exist. The cutting components of them are known as restriction endonuleases. The three
systems are known by roman numerals: Type I, II, and III restriction enzymes.
REBASE format 'cutzymes'(#15) lists enzyme type in its last field. The categories there do
not always match the the following short descriptions of the enzymes types. See
http://it.stlawu.edu/~tbudd/rmsyst.html for a better overview.
TypeI
Type I systems recognize a bipartite asymetrical sequence of 5-7 bp:
---TGA*NnTGCT--- * = methylation sites
---ACTNnA*CGA--- n = 6 for EcoK, n = 8 for EcoB
The cleavage site is roughly 1000 (400-7000) base pairs from the recognition site.
TypeII
The simplest and most common (at least commercially).
Site recognition is via short palindromic base sequences that are 4-6 base pairs long.
Cleavage is at the recognition site (but may occasionally be just adjacent to the
palindromic sequence, usually within) and may produce blunt end termini or staggered,
"sticky end" termini.
TypeIII
The recognition site is a 5-7 bp asymmetrical sequence. Cleavage is ATP dependent 24-26
base pairs downstream from the recognition site and usually yields staggered cuts 2-4
bases apart.
COMMENTS
I am trying to make this backwards compatible with Bio::Tools::RestrictionEnzyme.
Undoubtedly some things will break, but we can fix things as we progress.....!
I have added another comments section at the end of this POD that discusses a couple of
areas I know are broken (at the moment)
TO DO
• Convert vendors touse full names of companies instead of code
• Add regular expression based matching to vendors
• Move away from the archaic ^ notation for cut sites. Ideally I'd totally like to remove
this altogether, or add a method that adds it in if someone really wants it. We should
be fixed on a sequence, number notation.
FEEDBACK
Mailing Lists
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/wiki/Mailing_lists - About the mailing lists
Support
Please direct usage questions or support issues to the mailing list:
bioperl-l@bioperl.org
rather than to the module maintainer directly. Many experienced and reponsive experts will
be able look at the problem and quickly address it. Please include a thorough description
of the problem with code and data examples if at all possible.
Reporting Bugs
Report bugs to the Bioperl bug tracking system to help us keep track the bugs and their
resolution. Bug reports can be submitted via the web:
https://github.com/bioperl/bioperl-live/issues
AUTHOR
Rob Edwards, redwards@utmem.edu
CONTRIBUTORS
Heikki Lehvaslaiho, heikki-at-bioperl-dot-org Peter Blaiklock,
pblaiklo@restrictionmapper.org Mark A. Jensen, maj-at-fortinbras-dot-us
COPYRIGHT
Copyright (c) 2003 Rob Edwards.
Some of this work is 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.
SEE ALSO
Bio::Restriction::Analysis, Bio::Restriction::EnzymeCollection, Bio::Restriction::IO
APPENDIX
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.
new
Title : new
Function
Function : Initializes the Enzyme object
Returns : The Restriction::Enzyme object
Argument : A standard definition can have several formats. For example:
$re->new(-enzyme='EcoRI', -seq->'GAATTC' -cut->'1')
Or, you can define the cut site in the sequence, for example
$re->new(-enzyme='EcoRI', -seq->'G^AATTC'), but you must use a caret
Or, a sequence can cut outside the recognition site, for example
$re->new(-enzyme='AbeI', -seq->'CCTCAGC' -cut->'-5/-2')
Other arguments:
-isoschizomers=>\@list a reference to an array of
known isoschizomers
-references=>$ref a reference to the enzyme
-source=>$source the source (person) of the enzyme
-commercial_availability=>@companies a list of companies
that supply the enzyme
-methylation_site=>\%sites a reference to hash that has
the position as the key and the type of methylation
as the value
-xln_sub => sub { ($self,$cut) = @_; ...; return $xln_cut },
a coderef to a routine that translates the input cut value
into Bio::Restriction::Enzyme coordinates
( e.g., for withrefm format, this might be
-xln_sub => sub { length( shift()->string ) + shift } )
A Restriction::Enzyme object manages its recognition sequence as a Bio::PrimarySeq object.
The minimum requirement is for a name and a sequence.
This will create the restriction enzyme object, and define several things about the
sequence, such as palindromic, size, etc.
Essential methods
name
Title : name
Usage : $re->name($newval)
Function : Gets/Sets the restriction enzyme name
Example : $re->name('EcoRI')
Returns : value of name
Args : newvalue (optional)
This will also clean up the name. I have added this because some people get confused about
restriction enzyme names. The name should be One upper case letter, and two lower case
letters (because it is derived from the organism name, eg. EcoRI is from E. coli). After
that it is all confused, but the numbers should be roman numbers not numbers, therefore
we'll correct those. At least this will provide some standard, I hope.
site
Title : site
Usage : $re->site();
Function : Gets/sets 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
Side effect: the sequence is always converted to upper case.
The cut site can also be set by using methods cut and complementary_cut.
This will pad out missing sequence with N's. For example the enzyme Acc36I cuts at
ACCTGC(4/8). This will be returned as ACCTGCNNNN^
Note that the common notation ACCTGC(4/8) means that the forward strand cut is four
nucleotides after the END of the recognition site. The forward cut() in the coordinates
used here in Acc36I ACCTGC(4/8) is at 6+4 i.e. 10.
** This is the main setable method for the recognition site.
revcom_site
Title : revcom_site
Usage : $re->revcom_site();
Function : Gets/sets the complementary recognition sequence for the enzyme.
Example : $seq_string = $re->revcom_site();
Returns : String containing recognition sequence indicating
: cleavage site as in 'G^AATTC'.
Argument : none (sets on first call)
Throws : n/a
This is the same as site, except it returns the revcom site. For palindromic enzymes these
two are identical. For non-palindromic enzymes they are not!
On set, this also handles setting the revcom_recog attribute.
See also site above.
cut
Title : cut
Usage : $num = $re->cut(1);
Function : Sets/gets an integer indicating the position of cleavage
relative to the 5' end of the recognition sequence in the
forward strand.
For type II enzymes, sets the symmetrically positioned
reverse strand cut site by calling complementary_cut().
Returns : Integer, 0 if not set
Argument : an integer for the forward strand cut site (optional)
Note that the common notation ACCTGC(4/8) means that the forward strand cut is four
nucleotides after the END of the recognition site. The forward cut in the coordinates used
here in Acc36I ACCTGC(4/8) is at 6+4 i.e. 10.
Note that REBASE uses notation where cuts within symmetic sites are marked by '^' within
the forward sequence but if the site is asymmetric the parenthesis syntax is used where
numbering ALWAYS starts from last nucleotide in the forward strand. That's why AciI has a
site usually written as CCGC(-3/-1) actualy cuts in
C^C G C
G G C^G
In our notation, these locations are 1 and 3.
The cuts locations in the notation used are relative to the first (non-N) nucleotide of
the reported forward strand of the recognition sequence. The following diagram numbers the
phosphodiester bonds (marked by + ) which can be cut by the restriction enzymes:
1 2 3 4 5 6 7 8 ...
N + N + N + N + N + G + A + C + T + G + G + N + N + N
... -5 -4 -3 -2 -1
cuts_after
Title : cuts_after
Usage : Alias for cut()
complementary_cut
Title : complementary_cut
Usage : $num = $re->complementary_cut('1');
Function : Sets/Gets an integer indicating the position of cleavage
: on the reverse strand of the restriction site.
Returns : Integer
Argument : An integer (optional)
Throws : Exception if argument is non-numeric.
This method determines the cut on the reverse strand of the sequence. For most enzymes
this will be within the sequence, and will be set automatically based on the forward
strand cut, but it need not be.
Note that the returned location indicates the location AFTER the first non-N site
nucleotide in the FORWARD strand.
Read only (usually) recognition site descriptive methods
type
Title : type
Usage : $re->type();
Function : Get/set the restriction system type
Returns :
Argument : optional type: ('I'|II|III)
Restriction enzymes have been catezorized into three types. Some REBASE formats give the
type, but the following rules can be used to classify the known enzymes:
1. Bipartite site (with 6-8 Ns in the middle and the cut site is > 50 nt away) => type I
2. Site length < 3 => type I
3. 5-6 asymmetric site and cuts >20 nt away => type III
4. All other => type II
There are some enzymes in REBASE which have bipartite recognition site and cat far from
the site but are still classified as type I. I've no idea if this is really so.
seq
Title : seq
Usage : $re->seq();
Function : Get the Bio::PrimarySeq.pm object representing
: the recognition sequence
Returns : A Bio::PrimarySeq object representing the
enzyme recognition site
Argument : n/a
Throws : n/a
string
Title : string
Usage : $re->string();
Function : 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
recog
Title : recog
Usage : $enz->recog($recognition_sequence)
Function: Gets/sets the pure recognition site. Sets as
regexp if appropriate.
As for string(), the cut indicating carets (^)
are expunged.
Example :
Returns : value of recog (a scalar)
Args : on set, new value (a scalar or undef, optional)
revcom_recog
Title : revcom_recog
Usage : $enz->revcom_recog($recognition_sequence)
Function: Gets/sets the pure reverse-complemented recognition site.
Sets as regexp if appropriate.
As for string(), the cut indicating carets (^) are expunged.
Example :
Returns : value of recog (a scalar)
Args : on set, new value (a scalar or undef, optional)
revcom
Title : revcom
Usage : $re->revcom();
Function : Get a string representing the reverse complement of
: the recognition sequence.
Returns : String
Argument : n/a
Throws : n/a
recognition_length
Title : recognition_length
Usage : $re->recognition_length();
Function : Get the length of the RECOGNITION sequence.
This is the total recognition sequence,
inluding the ambiguous codes.
Returns : An integer
Argument : Nothing
See also: non_ambiguous_length
cutter
Title : cutter
Usage : $re->cutter
Function : Returns the "cutter" value of the recognition site.
This is a value relative to site length and lack of
ambiguity codes. Hence: 'RCATGY' is a five (5) cutter site
and 'CCTNAGG' a six cutter
This measure correlates to the frequency of the enzyme
cuts much better than plain recognition site length.
Example : $re->cutter
Returns : integer or float number
Args : none
Why is this better than just stripping the ambiguos codes? Think about it like this: You
have a random sequence; all nucleotides are equally probable. You have a four nucleotide
re site. The probability of that site finding a match is one out of 4^4 or 256, meaning
that on average a four cutter finds a match every 256 nucleotides. For a six cutter, the
average fragment length is 4^6 or 4096. In the case of ambiguity codes the chances are
finding the match are better: an R (A|T) has 1/2 chance of finding a match in a random
sequence. Therefore, for RGCGCY the probability is one out of (2*4*4*4*4*2) which exactly
the same as for a five cutter! Cutter, although it can have non-integer values turns out
to be a useful and simple measure.
From bug 2178: VHDB are ambiguity symbols that match three different nucleotides, so they
contribute less to the effective recognition sequence length than e.g. Y which matches
only two nucleotides. A symbol which matches n of the 4 nucleotides has an effective
length of 1 - log(n) / log(4).
is_palindromic
Title : is_palindromic
Alias : palindromic
Usage : $re->is_palindromic();
Function : Determines if the recognition sequence is palindromic
: for the current restriction enzyme.
Returns : Boolean
Argument : n/a
Throws : n/a
A palindromic site (EcoRI):
5-GAATTC-3
3-CTTAAG-5
is_symmetric
Title : is_symmetric
Alias : symmetric
Usage : $re->is_symmetric();
Function : Determines if the enzyme is a symmetric cutter
Returns : Boolean
Argument : none
A symmetric but non-palindromic site (HindI):
v
5-C A C-3
3-G T G-5
^
overhang
Title : overhang
Usage : $re->overhang();
Function : Determines the overhang of the restriction enzyme
Returns : "5'", "3'", "blunt" of undef
Argument : n/a
Throws : n/a
A blunt site in SmaI returns "blunt"
5' C C C^G G G 3'
3' G G G^C C C 5'
A 5' overhang in EcoRI returns "5'"
5' G^A A T T C 3'
3' C T T A A^G 5'
A 3' overhang in KpnI returns "3'"
5' G G T A C^C 3'
3' C^C A T G G 5'
overhang_seq
Title : overhang_seq
Usage : $re->overhang_seq();
Function : Determines the overhang sequence of the restriction enzyme
Returns : a Bio::LocatableSeq
Argument : n/a
Throws : n/a
I do not think it is necessary to create a seq object of these. (Heikki)
Note: returns empty string for blunt sequences and undef for ones that we don't know.
Compare these:
A blunt site in SmaI returns empty string
5' C C C^G G G 3'
3' G G G^C C C 5'
A 5' overhang in EcoRI returns "AATT"
5' G^A A T T C 3'
3' C T T A A^G 5'
A 3' overhang in KpnI returns "GTAC"
5' G G T A C^C 3'
3' C^C A T G G 5'
Note that you need to use method overhang to decide whether it is a 5' or 3' overhang!!!
Note: The overhang stuff does not work if the site is asymmetric! Rethink!
compatible_ends
Title : compatible_ends
Usage : $re->compatible_ends($re2);
Function : Determines if the two restriction enzyme cut sites
have compatible ends.
Returns : 0 if not, 1 if only one pair ends match, 2 if both ends.
Argument : a Bio::Restriction::Enzyme
Throws : unless the argument is a Bio::Resriction::Enzyme and
if there are Ns in the ovarhangs
In case of type II enzymes which which cut symmetrically, this function can be considered
to return a boolean value.
is_ambiguous
Title : is_ambiguous
Usage : $re->is_ambiguous();
Function : Determines if the restriction enzyme contains ambiguous sequences
Returns : Boolean
Argument : n/a
Throws : n/a
Additional methods from Rebase
is_prototype
Title : is_prototype
Usage : $re->is_prototype
Function : Get/Set method for finding out if this enzyme is a prototype
Example : $re->is_prototype(1)
Returns : Boolean
Args : none
Prototype enzymes are the most commonly available and usually first enzymes discoverd that
have the same recognition site. Using only prototype enzymes in restriction analysis
avoids redundancy and speeds things up.
is_neoschizomer
Title : is_neoschizomer
Usage : $re->is_neoschizomer
Function : Get/Set method for finding out if this enzyme is a neoschizomer
Example : $re->is_neoschizomer(1)
Returns : Boolean
Args : none
Neoschizomers are distinguishable from the prototype enzyme by having a different cleavage
pattern. Note that not all formats report this
prototype_name
Title : prototype_name
Alias : prototype
Usage : $re->prototype_name
Function : Get/Set method for the name of prototype for
this enzyme's recognition site
Example : $re->prototype_name(1)
Returns : prototype enzyme name string or an empty string
Args : optional prototype enzyme name string
If the enzyme itself is the prototype, its own name is returned. Not to confuse the
negative result with an unset value, use method is_prototype.
This method is called prototype_name rather than prototype, because it returns a string
rather than on object.
isoschizomers
Title : isoschizomers
Alias : isos
Usage : $re->isoschizomers(@list);
Function : Gets/Sets a list of known isoschizomers (enzymes that
recognize the same site, but don't necessarily cut at
the same position).
Arguments : A reference to an array that contains the isoschizomers
Returns : A reference to an array of the known isoschizomers or 0
if not defined.
This has to be the hardest name to spell, so now you can use the alias 'isos'. Added for
compatibility to REBASE
purge_isoschizomers
Title : purge_isoschizomers
Alias : purge_isos
Usage : $re->purge_isoschizomers();
Function : Purges the set of isoschizomers for this enzyme
Arguments :
Returns : 1
methylation_sites
Title : methylation_sites
Usage : $re->methylation_sites(\%sites);
Function : Gets/Sets known methylation sites (positions on the sequence
that get modified to promote or prevent cleavage).
Arguments : A reference to a hash that contains the methylation sites
Returns : A reference to a hash of the methylation sites or
an empty string if not defined.
There are three types of methylation sites:
• (6) = N6-methyladenosine
• (5) = 5-methylcytosine
• (4) = N4-methylcytosine
These are stored as 6, 5, and 4 respectively. The hash has the sequence position as the
key and the type of methylation as the value. A negative number in the sequence position
indicates that the DNA is methylated on the complementary strand.
Note that in REBASE, the methylation positions are given Added for compatibility to
REBASE.
purge_methylation_sites
Title : purge_methylation_sites
Usage : $re->purge_methylation_sites();
Function : Purges the set of methylation_sites for this enzyme
Arguments :
Returns :
microbe
Title : microbe
Usage : $re->microbe($microbe);
Function : Gets/Sets microorganism where the restriction enzyme was found
Arguments : A scalar containing the microbes name
Returns : A scalar containing the microbes name or 0 if not defined
Added for compatibility to REBASE
source
Title : source
Usage : $re->source('Rob Edwards');
Function : Gets/Sets the person who provided the enzyme
Arguments : A scalar containing the persons name
Returns : A scalar containing the persons name or 0 if not defined
Added for compatibility to REBASE
vendors
Title : vendors
Usage : $re->vendor(@list_of_companies);
Function : Gets/Sets the a list of companies that you can get the enzyme from.
Also sets the commercially_available boolean
Arguments : A reference to an array containing the names of companies
that you can get the enzyme from
Returns : A reference to an array containing the names of companies
that you can get the enzyme from
Added for compatibility to REBASE
purge_vendors
Title : purge_vendors
Usage : $re->purge_references();
Function : Purges the set of references for this enzyme
Arguments :
Returns :
vendor
Title : vendor
Usage : $re->vendor(@list_of_companies);
Function : Gets/Sets the a list of companies that you can get the enzyme from.
Also sets the commercially_available boolean
Arguments : A reference to an array containing the names of companies
that you can get the enzyme from
Returns : A reference to an array containing the names of companies
that you can get the enzyme from
Added for compatibility to REBASE
references
Title : references
Usage : $re->references(string);
Function : Gets/Sets the references for this enzyme
Arguments : an array of string reference(s) (optional)
Returns : an array of references
Use purge_references to reset the list of references
This should be a Bio::Biblio object, but its not (yet)
purge_references
Title : purge_references
Usage : $re->purge_references();
Function : Purges the set of references for this enzyme
Arguments :
Returns : 1
clone
Title : clone
Usage : $re->clone
Function : Deep copy of the object
Arguments : -
Returns : new Bio::Restriction::EnzymeI object
This works as long as the object is a clean in-memory object using scalars, arrays and
hashes. You have been warned.
If you have module Storable, it is used, otherwise local code is used. Todo: local code
cuts circular references.
_expand
Title : _expand
Function : Expand nucleotide ambiguity codes to their representative letters
Returns : The full length string
Arguments : The string to be expanded.
Stolen from the original RestrictionEnzyme.pm