man Bio::Restriction::Enzyme (3): A single restriction endonuclease

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.

  [email protected]                  - General discussion
  http://bioperl.org/wiki/Mailing_lists  - About the mailing lists

Support

Please direct usage questions or support issues to the mailing list:

[email protected]

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, [email protected]

CONTRIBUTORS

Heikki Lehvaslaiho, heikki-at-bioperl-dot-org Peter Blaiklock, [email protected] Mark A. Jensen, maj-at-fortinbras-dot-us

COPYRIGHT

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.

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

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

SYNOPSIS

DESCRIPTION

RESTRICTION MODIFICATION SYSTEMS

TypeI

TypeII

TypeIII

COMMENTS

TO DO

FEEDBACK

Mailing Lists

Support

Reporting Bugs

AUTHOR

CONTRIBUTORS

COPYRIGHT

APPENDIX

new

Essential methods

name

site

revcom_site

cut

cuts_after

complementary_cut

Read only (usually) recognition site descriptive methods

type

seq

string

recog

revcom_recog

revcom

recognition_length

cutter

is_palindromic

is_symmetric

overhang

overhang_seq

compatible_ends

is_ambiguous

Additional methods from Rebase

is_prototype

is_neoschizomer

prototype_name

isoschizomers

purge_isoschizomers

methylation_sites

purge_methylation_sites

microbe

source

vendors

purge_vendors

vendor

references

purge_references

clone

_expand

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