Check
Oligonucleotides against Database
In this step all the oligos identified so far are screened for their
ability to target the identified target group to the exclusion of all
other records. Potentially useful oligos will be those which
target the most records within the defined target group whilst
targeting the least number of records outside of this target group.
1) On the main panel, click '
Check Oligonucleotides against
Database'. A dialog box is displayed , showing the database
records (top left panel), with target records marked; a list of those
target records used to design the oligonucleotides to be screened (top
right table); and a summary of the number of oligonucleotides
identified previously.
2) Select the maximum number of acceptable non-target hits you want
your
oligonucleotide to target.
3) Click '
Search Database Records' to start analysis. The speed of
this step will depend on the number of oligos to be screened, the size
of the database and the maximum number of non-targets allowed.
Each oligo is screened against each database record and the number of
matches (hits) recorded - oligos which exceed the maximum number of
non-target records hit are discarded as the analysis proceeds, as
indicated by the number display near the bottom of the
dialog.
As more records are excluded, the program will
speed
up, so the estimation of the time remaining, appearing at the bottom of
the dialog box, will be somewhat conservative initially. The
search can be aborted at any time by clicking the '
Abort
search'
button.
4) Analysis complete, the successful oligos are listed in the main
table. Oligos are listed as 5' - 3' sense sequences, colour coded
(
,
,
) according
to their success in terms of targeting the specified target
group.
Alongside each oligo is:
- Total number of database records hit.
- Of these, total number of target records hit.
- Total number of target records hit as a percentage of the target
group.
- Total number of non-target records hit.
- Total number of non-target records hit as a percentage of all
non-target records.
- Melting temperature - calculated by the Wallace Rule ().
- % G-C content.
- Number of degenerate bases in oligonucleotide.
Click on column header to sort any colum in ascending numerical (or
alphabetical for oligos) order.
Any oligonucleotide listed can be explored further by double clicking
on its
sequence. This opens a further dialog
box displaying the selected
oligonucleotide and the database, with targets marked.