Now that you have a sequence, you can design the oligos. The defaults are 60bp oligos with 20bp overlaps. These values work well for us with yeast and mammalian sequences which are ~40% GC. You may want to play with these values if your sequences are outside of this range. GeneDesign will break the sequence into roughly 500bp chunks on unique restriction sites (restriction sites must be non-blunt, non-1bp overhang cutters that have cleavage sites internal to the recognition sequence) and then break those chunks into an even number of 60bp oligos. The oligos are then adjusted for even lengths and equal melting temperatures.
The Oligo Selection Module - A Sample
Every 500bp chunk is displayed on the oligo summary page. For each chunk, the order number, length in bp, average
Tm of overlaps, and 3' unique site appear in that order in the vertical bar at left.
The chunk sequence and the sequences of oligos are printed for visual confirmation of appropriate alignment at the
top of each chunk. Information for each oligo is contained in rows under the line up. This information includes
oligo length, start and stop coordinates, and sense. It also includes
data on the length and melting temperatures of the 5' and 3' overlaps. The 5' to 3' sequence of each oligo is printed at the far right.
Melting temperature is calculated in three
ways. The first column uses the formula from Baldino
et al. This is the same
formula used by Strider. The second column uses a derivative
of that formula, as used in Primer3.
The third column uses the nearest neighbor (Borer
et al) parameters for DNA/DNA
duplexes as defined in Sugimoto et al.
These parameters are used in the formula below, derived from Rychlik
et al, where ΔH
is enthalpy, ΔS
is entropy, R is the molar gas constant, (16.6*log[Na+]) is the salt correction,
and 3.4 kcal/mol is the activation energy required to go from single to double
stranded DNA.
If there are no further adjustments to be made, you can export these oligos to a delineated text file for ordering. If you select a designation string for the oligos and a delineator, you can use the "Order Form" button to kick out a list that can easily be opened in Excel or sent to the oligo producing company of your choice. The oligos will be numbered from first chunk to last, using the designation string. For example, if your designation string is "syngene", the first oligo will be called "syngene01" and the next will be "syngene02", etc.
The "Full Report" button will open in a new window a summary of all the steps you took in the Design a Gene path, from Reverse Translation to Oligo Designs.