SYNOPSIS

# do not run this class directly

DESCRIPTION

This module defines methods for a single restriction endonuclease. For an implementation, see Bio::Restriction::Enzyme.

FEEDBACK

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Reporting Bugs

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AUTHOR

Heikki Lehvaslaiho, heikki-at-bioperl-dot-org

CONTRIBUTORS

Rob Edwards, [email protected]

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.

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  : 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.

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

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

non_ambiguous_length

 Title     : non_ambiguous_length
 Usage     : $re->non_ambiguous_length();
 Function  : Get the nonambiguous length of the RECOGNITION sequence.
             This is the total recognition sequence,
             excluding 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 ambiguous 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
 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.

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:

• (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 or Bio::Annotation::Reference 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.