]> Simple version of primer3 to pick PCR primers (EMBOSS) nucleic:primers http://bioweb.pasteur.fr/docs/EMBOSS/primers.html primers &emboss_init; input Input section sequence sequence -- dna [sequences] (-sequence) perl " -sequence=$value -sformat=fasta" 1 The sequence from which to choose primers. The sequence must be presented 5' -> 3' dna 8 seqsfile perl 1 required Required section target Target region(s) (-target) perl " -target=$value" 2 If one or more Targets is specified then a legal primer pair must flank at least one of them. A Target might be a simple sequence repeat site (for example a CA repeat) or a single-base-pair polymorphism. The value should be a space-separated list of <BR> <start>,<end> <BR> pairs where <start> is the index of the first base of a Target, and <end> is the last <BR> E.g. 50,51 requires primers to surround the 2 bases at positions 50 and 51. advanced Advanced section minsize Primer minimum size (-minsize) 18 perl (defined $value && $value != $vdef)? " -minsize=$value" : "" 3 Minimum acceptable length of a primer. Must be greater than 0 and less than or equal to MAX-SIZE. 1 maxsize Primer maximum size (-maxsize) 27 perl (defined $value && $value != $vdef)? " -maxsize=$value" : "" 4 Maximum acceptable length (in bases) of a primer. Currently this parameter cannot be larger than 35. This limit is governed by the maximum oligo size for which Primer3's melting-temperature is valid. acd $MinSize 35 maxdifftm Maximum difference in Tm of primers (-maxdifftm) 100.0 perl (defined $value && $value != $vdef)? " -maxdifftm=$value" : "" 5 Maximum acceptable (unsigned) difference between the melting temperatures of the forward and reverse primers. output Output section explainflag Explain flag (-explainflag) 0 perl ($value)? " -explainflag" : "" 6 If this flag is non-0, produce LEFT-EXPLAIN and RIGHT-EXPLAIN, output, which are intended to provide information on the number of primer pairs that Primer3 examined, and statistics on the number discarded for various reasons. numreturn Number of results to return (-numreturn) 5 perl (defined $value && $value != $vdef)? " -numreturn=$value" : "" 7 The maximum number of primer pairs to return. Primer pairs returned are sorted by their 'quality', in other words by the value of the objective function (where a lower number indicates a better primer pair). Caution: setting this parameter to a large value will increase running time. outfile outfile (-outfile) outfile.out perl " -outfile=$value" 8 auto perl " -auto -stdout" 9