The selector procedure involves digesting genomic DNA with restriction enzymes, to form fragments that can then be circularized with the aid of selector probes. Therefore, a fundamental part of selector design is the in silico digestion.
A selector assay can include multiple restriction digestion reactions, and each reaction can contain one or more restriction enzymes. The goal of this procedure is to generate fragments that are of the appropriate size for selection, and to do so for all targeted regions at the same time.
Disperse makes use of PieceMaker to perform the digestion. The set of restriction reactions to use is specified by the "piecemaker.reactionFile" entry in the settings file. This file defines a set of restriction enzymes, and specifies which combinations of one or more enzymes to use as restriction reactions in the digestion.
The file can be provided either in plain text format,or in an xml format. Disperse will check the file name of the reaction file. If it ends with '.xml' it will be parsed as an xml file, otherwise it will be parsed as a plain text file.
Fields are separated by tabs, lines beginning with # are comments. The recognition sequence is in 5' to 3', and the numbers specify the position where cleavage occurs on the top (same as sequence) and bottom (complement of sequence) strand, where 0 is the position immediately upstream of the first base in the sequence. Don't mind the 0's. They need to be there, but are not used by Disperse.
#Reaction list stored Thu Dec 20 11:19:59 CET 2007 [Enzymes] Bbv I 0 0 0 GCAGC;13;17 GCTGC;-12;-8 Mnl I 0 0 0 CCTC;11;10 GAGG;-6;-7 Mse I 0 0 0 TTAA;1;3 Sty I 0 0 0 CCWWGG;1;5 Alu I 0 0 0 AGCT;2;2 [Reactions] Alu I Bbv I Sty I Mnl I Sty I Mse I
<pi:reactions xmlns:pi="http://genefront.net/piecemaker">
<pi:enzymeList>
<pi:enzyme pi:name="Bbv I">
<pi:cutPattern pi:sequence="GCAGC" pi:cutPositionTop="13"
pi:cutPositionBottom="17" />
<pi:cutPattern pi:sequence="GCTGC" pi:cutPositionTop="-12"
pi:cutPositionBottom="-8" />
</pi:enzyme>
<pi:enzyme pi:name="Mse I">
<pi:cutPattern pi:sequence="TTAA" pi:cutPositionTop="1"
pi:cutPositionBottom="3" />
</pi:enzyme>
<pi:enzyme pi:name="Sty I">
<pi:cutPattern pi:sequence="CCWWGG" pi:cutPositionTop="1"
pi:cutPositionBottom="5" />
<pi:cutPattern pi:sequence="CCSSGG" pi:cutPositionTop="1"
pi:cutPositionBottom="5" />
</pi:enzyme>
<pi:enzyme pi:name="Alu I">
<pi:cutPattern pi:sequence="AGCT" pi:cutPositionTop="2"
pi:cutPositionBottom="2" />
</pi:enzyme>
<pi:enzyme pi:name="Mnl I">
<pi:cutPattern pi:sequence="CCTC" pi:cutPositionTop="11"
pi:cutPositionBottom="10" />
<pi:cutPattern pi:sequence="GAGG" pi:cutPositionTop="-6"
pi:cutPositionBottom="-7" />
</pi:enzyme>
</pi:enzymeList>
<pi:reactionList>
<pi:reaction pi:name="Alu I">
<pi:enzyme pi:name="Alu I" />
</pi:reaction>
<pi:reaction pi:name="Bbv I">
<pi:enzyme pi:name="Bbv I" />
</pi:reaction>
<pi:reaction pi:name="Sty I/Mnl I">
<pi:enzyme pi:name="Sty I" />
<pi:enzyme pi:name="Mnl I" />
</pi:reaction>
<pi:reaction pi:name="Sty I/Mse I">
<pi:enzyme pi:name="Sty I" />
<pi:enzyme pi:name="Mse I" />
</pi:reaction>
</pi:reactionList>
</pi:reactions>