primer3 multiple -excludedregion problem

Question

[gogo.gautier]

Hello,

First posting here!

I am having a problem running primer3 (actually using EMBOSS eprimer32) with the option to exclude regions where primer should not overlap. I used the option -excludedregion. It seems to work for a single region but not a multiple region. For example:

eprimer32 -sequence input.fasta \
  -prange 170-250 \
  -excludedregion 345,360 \
  -targetregion 180,335 \
  -maxdifftm 1 \
  -gcclamp 1 \
  -opttm 58 \
  -maxtm 60 \
  -mintm 54 \
  -optsize 20 \
  -minsize 17 \
  -maxsize 24 \
  -outfile output.primer \
  -numreturn 1 \
  -mingc 30 \
  -maxgc 70

does seem to work with primer designed at position 142-162 and 361-381 BUT the following with 2 regions to avoid:

eprimer32 -sequence input.fasta \
  -prange 170-250 \
  -excludedregion 20,25 345,360 \
  -targetregion 180,335 \
  -maxdifftm 1 \
  -gcclamp 1 \
  -opttm 58 \
  -maxtm 60 \
  -mintm 54 \
  -optsize 20 \
  -minsize 17 \
  -maxsize 24 \
  -outfile output.primer \
  -numreturn 1 \
  -mingc 30 \
  -maxgc 70

is not working and primers are designed at position 142-162 and 340-360 , the second primer overlapping with my excluded region.

Any idea what I'm doing wrong?

Many thanks for any help or pointers!

Philippe

PS: Help file for the option is saying:

-excludedregion     range      [(full sequence)] Primer oligos may not
                                  overlap any region specified in this tag.
                                  The associated value must be a
                                  space-separated list of
                                  (start),(end)
                                  pairs where (start) is the index of the
                                  first base of the excluded region, and and
                                  (end) is the last. This tag is useful for
                                  tasks such as excluding regions of low
                                  sequence quality or for excluding regions
                                  containing repetitive elements such as ALUs
                                  or LINEs.
                                  E.g. 401,407 68,70 forbids selection of
                                  primers in the 7 bases starting at 401 and
                                  the 3 bases at 68.