Find
Oligonucleotides.
The database
constructed, and target taxon
identified, the next step is to identify all possible
oligonucleotides that could
describe the selected group. At this stage we are only
interested in
identifying oligonucleotides shared by as many of
the sequences in the target group as possible. Later, these
potentially useful oligonucleotides will be screened for their ability
to exclude
non-target records.
1) Select 'Find Oligonucleotides' from the main window. Records
within the target group (
) are displayed in
the dialog box table:
2) Using the check boxes in the right-most
table column
,
select representatives of the target group from which to
generate/design the oligonucleotides.
If the selected target
group is small (e.g. < 10) selecting all will probably be the best
strategy. However, with larger target groups it may be more
productive to select only a proportion (say, no more than 15), as too
large a selection may resulting in the program missing
oligonucleotides, which whilst able to describe every single
target record, may not be effective in descringuishing non-targets
fronm targets (choosing ~15 ensures a compromise between finding the
best oligonucleotide which describes the target group, and one which
exclused the most non-target records).
At least one target record must be selected to design from, for the
program to continue.
3) Use the 'Required length' spinner box to select the required oligo
length (between 3 and 100 nucleotides). Use the 'Maximum
degenerate bases' spinner box to select the maximum
number of acceptable degenerate bases.
4) Once you are happy with your selection, click the '
Find
Oligonucleotides' button. The program will then collect together
the selected sequences, align the sequences (if necessary) and generate
a consensus sequence from which the oligonucleotides are then
generated. Once complete, the total number of putative
oligos identified is quoted along with a breakdown of how many have
each sort of degeneracy.
5) If you are happy with the number of oligos generated, click '
Accept' to
proceed to the next stage. '
Reset' buttons clears all selections.
'
Cancel' returns to the main panel without accepting oligos. Tip:
the speed of the next stage of the program is partially dependent on
the number of putative oligos identifed. Thus, if a very large
number of oligos are identifed at this stage (e.g. >700) it might be
better to amend parameter choices (i.e., reduce maximum degeneracy;
select more target records to design from) to reduce this figure, to a
more managable level.