Bio::Restriction::EnzyUser)Contributed Perl DocumenBio::Restriction::Enzyme(3)NAMEBio::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:
http://bugzilla.open-bio.org/
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
forwad 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
perl v5.14.1 2011-07-22 Bio::Restriction::Enzyme(3)
