GeneDesign object that represents a type II restriction enzyme
Sarah Richardson <SMRichardson@lbl.gov>
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.
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
-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()
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.
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.
Given a nucleotide sequence context, what overhang does this enzyme leave, and how far away from the cutsite is it?
Generates a tab delimited display string that can be used to print enzyme information out in a tabular format.
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
Returns a buffer in which both enzymes will have at least a thresholded amount of activity.
Returns the number of units needed to cleave some 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.
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.
Arguments : an arrayref of vendor abbreviations; see vendor(). Returns : 1 if the enzyme is supplied by any of the vendors queried, 0 else.
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.
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.
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.
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.
Arguments : 1 if star activity required, 0 else Returns : 1 if the enzyme meets the star activity requirements, 0 else.
Arguments : an arrayref of acceptable integer incubation temperatures Returns : 1 if the enzyme meets the incubation temperature requirements, 0 else.
Arguments : an acceptable integer inactivation temperature maximum Returns : 1 if the enzyme meets the inactivation temperature requirement, 0 else.
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.
Arguments : an arrayref of acceptable recognition site lengths Returns : 1 if the enzyme meets the recognition site length requirements, 0 else.
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.
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.
Methods for setting and accessing enzyme attributes
The name of the enzyme.
The name of the enzyme.
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.
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.
The length in bases of the recognition sequence (recseq).
The effect of CpG methylation on the enzyme's efficacy.
The effect of Dcm methylation on the enzyme's efficacy.
The effect of Dam methylation on the enzyme's efficacy.
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.
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
The temperature in degrees Celsius that deactivates the enzyme.
The time required at inactivation temperature to deactivate the enzyme.
Incubation temperature for the best enzyme activity, in degrees Celsius.
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.
Synonym for recseq
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.
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 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.
Short cut to accessing class regular expressions
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.
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.
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.
Information about the overhang the enzyme leaves.
pal = palindromic nonpal = nonpalindromic pnon = potentially nonpalindromic, or sometimes palindromic and sometimes nonpalindromic unknown = unknown
1 if the enzyme exhibits star activity, 0 else
The offset in nucleotides of the enzymes recognition site in an ORF
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