Bio::Restriction::Enzyme - A single restriction endonuclease (cuts DNA at specific locations) # 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=new Bio::Restriction::Enzyme (-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"; 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. 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. 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) 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.

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/MailList.shtml - About the mailing lists

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 email or the web: bioperl-bugs@bio.perl.org http://bugzilla.bioperl.org/ Rob Edwards, redwards@utmem.edu Heikki Lehvaslaiho, heikki@ebi.ac.uk Peter Blaiklock, pblaiklo@restrictionmapper.org 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. Bio::Restriction::Analysis, Bio::Restriction::EnzymeCollection, Bio::Restriction::IO 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 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.

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 : Sequence of the site 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! 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

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.

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: Bipartite site (with 6-8 Ns in the middle and the cut site is > 50 nt away) => type I Site length < 3 => type I 5-6 asymmetric site and cuts >20 nt away => type III 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

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.

is_palindromic
Title : is_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

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 restriciton analysis avoids redundacy and speeds things up.

prototype_name
Title : prototype_name 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 protype, 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 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. Added for compatibility to REBASE

purge_isoschizomers
Title : purge_isoschizomers 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: 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.