/ 
Bio-GeneDesign-5.56
River stage zero No dependents
/ Bio::GeneDesign::RestrictionEnzyme

NAME

Bio::GeneDesign::RestrictionEnzyme

VERSION

Version 5.56

DESCRIPTION

GeneDesign object that represents a type II restriction enzyme

AUTHOR

Sarah Richardson <SMRichardson@lbl.gov>

CONSTRUCTOR METHODS

new

You can create a new enzyme or clone an existing enzyme to create a new instance of an abstract enzyme definition. To do this, provide the -enzyme flag; the constructor will ignore every other argument except for -start.

Required arguments:

EITHER

    -enzyme : a Bio::GeneDesign::RestrictionEnzyme object to clone

OR
    -id     : The name of the enzyme (i.e., BamHI)
    -cutseq : The string describing the enzyme's recognition and cleavage
              site

Optional arguments:

-temp     : The incubation temperature for the enzyme
-tempin   : The heat inactivation temperature for the enzyme
-score    : A float score, usually the price of the enzyme in dollars
-methdam  : Sensitivity to dam methylation; can take the values
              b or blocked,
              i or inhibited,
              u or unknown,
            if undefined, will take the value indifferent.
-methdcm  : Sensitivity to dcm methylation; can take the values
              b or blocked,
              i or inhibited,
              u or unknown,
            if undefined, will take the value indifferent.
-methcpg  : Sensitivity to cpg methylation; can take the values
              b or blocked,
              i or inhibited,
              u or unknown,
            if undefined, will take the value indifferent.
-vendors  : a string of single letter codes that represent vendor
            availability - no spaces.  see vendor() for a list of the
            codes.
-staract  : Whether or not the enzyme exhibits star activity - 1 or 0.
-buffers  : a hash reference; keys are buffer names and values are the
            enzyme activity in that buffer. For example:
            NEB1 => 50, NEB2 => 100, etc.
-start    : An integer representing an offset; usually used only in
            cloned instances, as opposed to abstract instances.
-exclude  : An arrayref full of ids for enzymes that should be
            considered mutually exclusive to this enzyme - see exclude()

FUNCTIONAL METHODS

clone

By default in GeneDesign code, RestrictionEnzyme objects are meant to stand as abstracts - that is, they stand for BamHI in general, and not for a particular instance of a BamHI recognition site. If you want to use the objects in the latter sense, you will need to clone the abstract object instantiated when the definition file is read in, thus generating an arbitrary number of BamHI instances that can then be differentiated by their start attributes.

positions

Generates a hash describing the positions of the enzyme's recognition sites in a nucleotide sequence. Keys are offset in nucleotides, and values are the recognition site found at said offset as a string.

overhang

Given a nucleotide sequence context, what overhang does this enzyme leave, and how far away from the cutsite is it?

Arguments:

display

Generates a tab delimited display string that can be used to print enzyme information out in a tabular format.

common_buffers

Returns an array reference listing the buffers, if any, in which two enzymes both have 100% activity. in boolean mode returns the number of buffers

acceptable_buffer

Returns a buffer in which both enzymes will have at least a thresholded amount of activity.

units

Returns the number of units needed to cleave some sequence

FILTERING METHODS

filter_by_sequence

Arguments: an arrayref of string nucleotide sequences (may be ambiguous)
           a flag indicating whether or not the sequences in the array are
            required (1 means they must NOT match; default 0 means they must
            match)

Returns : 1 if the enzyme passes;
          0 if the enzyme fails.

filter_by_score

Arguments : a float

Returns   : 1 if the enzyme's score is less than or equal to the argument,
            0 if the enzyme's score is higher.

filter_by_vendor

Arguments : an arrayref of vendor abbreviations; see vendor().

Returns   : 1 if the enzyme is supplied by any of the vendors queried,
            0 else.

filter_by_buffer_activity

Arguments : a hashref of buffer thresholds; the key is the buffer name, the
              value is an activity threshold.

Returns   : 1 if the enzyme meets all the buffer requirements,
            0 else.

filter_by_dcm_sensitivity

Arguments : an arrayref of sensitivity values; the key is the sensitivity
              blocked, inhibited, or indifferent

Returns   : 1 if the enzyme meets the dcm sensitivity requirements,
            0 else.

filter_by_dam_sensitivity

Arguments : an arrayref of sensitivity values; the key is the sensitivity
              blocked, inhibited, or indifferent

Returns   : 1 if the enzyme meets the dam sensitivity requirements,
            0 else.

filter_by_cpg_sensitivity

Arguments : an arrayref of sensitivity values; the key is the sensitivity
              blocked, inhibited, or indifferent

Returns   : 1 if the enzyme meets the cpg sensitivity requirements,
            0 else.

filter_by_star_activity

Arguments : 1 if star activity required, 0 else

Returns   : 1 if the enzyme meets the star activity requirements,
            0 else.

filter_by_incubation_temperature

Arguments : an arrayref of acceptable integer incubation temperatures

Returns   : 1 if the enzyme meets the incubation temperature requirements,
            0 else.

filter_by_inactivation_temperature

Arguments : an acceptable integer inactivation temperature maximum

Returns   : 1 if the enzyme meets the inactivation temperature requirement,
            0 else.

filter_by_base_ambiguity

Arguments : "nonNonly" if any non N bases are allowed; "ATCGonly" if only
              A, T, C, or G are allowed

Returns   : 1 if the enzyme meets the ambiguous nucleotide requirement,
            0 else.

filter_by_length

Arguments : an arrayref of acceptable recognition site lengths

Returns   : 1 if the enzyme meets the recognition site length requirements,
            0 else.

filter_by_overhang_palindromy

Arguments : an arrayref of acceptable overhang palindromys, from the list
              pal (palindromic),
              nonpal (nonpalindromic),
              pnon (potentially nonpalindromic)

Returns   : 1 if the enzyme meets the palindromy requirements,
            0 else.

filter_by_stickiness

Arguments : an arrayref of acceptable overhang orientations, from the list
              1 (single basepair overhang),
              5 (five prime overhang),
              3 (three prime overhang),
              b (blunt ended)

Returns   : 1 if the enzyme meets the overhang requirements,
            0 else.

ACCESSOR METHODS

Methods for setting and accessing enzyme attributes

id

The name of the enzyme.

display_name

The name of the enzyme.

score

This attribute initially holds the price in dollars per unit of the enzyme (2009 US Dollars) but can be used to hold any score or cost value.

aggress

Aggressiveness is the number of recognition sites in a template piece of DNA (usually lambda, but sometimes adeno2, pBR322, pUC19, pXba, etc) over the total length of that template piece of DNA. This number tells the manufacturer how much enzyme to sell as a "unit" - the amount of enzyme required to fully digest one microgram of template DNA under reaction conditions in an hour.

len

The length in bases of the recognition sequence (recseq).

methcpg

The effect of CpG methylation on the enzyme's efficacy.

methdcm

The effect of Dcm methylation on the enzyme's efficacy.

methdam

The effect of Dam methylation on the enzyme's efficacy.

buffers

A hash reference where the keys are buffer names and the values are the activity level of the enzyme in that Buffer. Since most of the enzymes in the default GeneDesign list are NEB enzymes, this is usually full of NEB buffers.

vendors

A hash reference where the keys are abbreviations for and the values are names of vendors that stock the enzyme. These are read in from the enzyme file.

B = Invitrogen
C = Minotech
E = Stratagene
F = Thermo Scientific Fermentas
I = SibEnzyme
J = Nippon Gene Co.
K = Takara
M = Roche Applied Science
N = New England Biolabs
O = Toyobo Technologies
Q = Molecular Biology Resources
R = Promega
S = Sigma Aldrich
U = Bangalore Genei
V = Vivantis
X = EURx
Y = CinnaGen

tempin

The temperature in degrees Celsius that deactivates the enzyme.

timein

The time required at inactivation temperature to deactivate the enzyme.

temp

Incubation temperature for the best enzyme activity, in degrees Celsius.

recseq

This attribute is the "clean" description of the enzyme's recognition sequence - that is, no information about cleavage site can be gained from this attribute. This is determined automatically from the cleavage string (cutseq) at instantiation.

seq

Synonym for recseq

cutseq

This attribute is the string description of the enzyme's recognition sequence. It includes information about both the recognition and cleavage sites. See http://rebase.neb.com/rebase/rebrec.html for help interpreting this field.

regex

This attribute stores a set of regular expressions as an array reference to speed the search for recognition sites in sequence. The first entry in the arrayref is the regular expression representing the forward orientation of the recognition sequence; the second entry represents the reverse orientation and is only defined if the recognition site is nonpalindromic.

This attribute is defined at instantiation.

class

Class describes the cutting behavior of an enzyme. The classes used by GeneDesign uses a generalized subset of the classes as described at Rebase - for the purposes of enzyme editing, three classes have so far proven to be enough. See http://rebase.neb.com/cgi-bin/sublist for the full description of enzyme classes.

IIP : This enzyme has a symmetric target and a symmetric cleavage site; this
      usually means that the enzyme cleaves inside its own recognition site.
      This is not the same as overhang palindromy!

IIA : This enzyme has an asymmetric recognition site and usually cleaves
      outside of it.

IIB : This enzyme has one recognition site and two cleavage sites, one on
      either side of the recognition site, and thus cuts itself out of
      sequence.

classex

class_regexes

Short cut to accessing class regular expressions

type

Type describes the kind of overhang left by an enzyme. This is probably not a good use of the word type.

Type may be 5', for a five prime overhang; 3', for a three prime overhang; or b for blunt ends.

onebpoverhang

One basepair overhangs can be harder to ligate than blunt ends. This attribute returns 1 if an enzyme leaves a 1bp overhang and 0 else.

exclude

Some enzymes share overlapping recognition sites. If you are trying to ensure the absence or uniqueness of a recognition site, you will want to be sure to exclude isoschizomers and neoschizomers from consideration elsewhere. The exclude attribute stores an array reference that lists the ids of neo- and isoschizomers - or any arbitrary enzyme that is incompatible with this enzyme - for easy lookup.

palindromy

Information about the overhang the enzyme leaves.

pal     = palindromic
nonpal  = nonpalindromic
pnon    = potentially nonpalindromic, or sometimes palindromic and sometimes
          nonpalindromic
unknown = unknown

staract

1 if the enzyme exhibits star activity, 0 else

start

The offset in nucleotides of the enzymes recognition site in an ORF

outside_cut

inside_cut

COPYRIGHT AND LICENSE

Copyright (c) 2015, Sarah Richardson All rights reserved.

Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met:

* Redistributions of source code must retain the above copyright notice, this list of conditions and the following disclaimer.

* Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution.

* The names of Johns Hopkins, the Joint Genome Institute, the Lawrence Berkeley National Laboratory, the Department of Energy, and the GeneDesign developers may not be used to endorse or promote products derived from this software without specific prior written permission.

THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS "AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE DEVELOPERS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.