GeneDesign's Silent Site Insertion module allows you to process a nucleotide sequence for modular mutagenesis. You can choose the vector you plan to use to carry your synthetic gene, or you can define a list of restriction enzymes to be considered for insertion. Vector choice is an extremely important step in the process. The purpose is to instruct the program on what restriction sites you DO NOT WANT to use as special "landmark restriction sites" in your synthetic sequence, namely those already present in the vector. If you choose a very large vector with many sites in it, the program will not have any sites left to play with. Therefore, if you have a choice, use the smallest vector available to you. In practice pUC18/19 work well; derivatives lacking the multiple cloning site work even better.
The Silent Site Insertion Module - Vector Selection
Next you must choose the sites to be inserted. If you specify a vector sequence, all sites in the vector are automatically removed from consideration as landmark restriction sites by the program. There are three ways to choose eligible sites. You may (A) simply select the sites from a list. Click on the site name in the left column and then the arrow button at the bottom of the column to move that site to the right hand column. Repeat the process on the right side to remove it. Only sites in the right column will be considered. Or you may (B) select the name of the vector you will be using from a pull-down menu. GeneDesign has the sequences of these vectors and will determine which enzymes are absent from this vector automatically for you. Lastly, you may (C) provide a vector sequence and a name for that vector. GeneDesign will parse the sequence and determine which enzymes are absent from your vector.
You may in addition specify a list of enzymes that must not appear in the
final sequence. (D) Click on the site name in the left column
and then the arrow button at the bottom of the column to move that site to
the right hand column.
Repeat the process on the right side to remove it. Only sites in the right
column will be processed. The sequence will be searched for these sites and
if they are found in your first-pass synthetic sequence, they will be removed. These
sites will not be considered for landmark restriction sites.
At (E) you can define the ranking criteria for sites to
be inserted. We have found the default criteria to be more than adequate for
most purposes. For more information about ranking enzymes, see Enzyme
Choosing.
Once
you have selected the vector and/or the sites you can click "Next
Step"
to move on to the next step in site
selection.
The Silent Site Insertion Module - Landmark Selection
Easiest method: You can have the computer select sites for you with the "Pick Sites For Me" button. Enter an amino acid interval and GeneDesign will select sites on that interval using the criteria you defined in the previous screen. The program's choices will be presented to you using the same screen and you will have a chance to edit them. You can change the amino acid interval and have the program reconsider as many times as you like.
Manual method: You can make your own landmark selections. Each pulldown menu is anchored by a line and a dot between a pair of amino acids; these only appear where a silent mutation is possible. The pulldown menus are populated with lists of enzymes that can be introduced at those positions. A black line indicates that none of the enzymes in the list are absent from the vector you selected (or present in the list of sites you created). A red line indicates that there are one or more enzymes in the list that are absent from the vector.
If you have the computer select the sites it will use blue lines to indicate its selections.
Silent Site Insertion Module - Summary of Sites
Once you are satisfied with the sites that have been selected you can click the "Continue to Summary" button. The next screen will provide you with a detailed list of the sites chosen for silent insertion. The first column is the site name. The next column is the recognition sequence, followed by an asterisk if the enzyme exhibits star activity. The third column is the nucleotide position of the site. The fourth column defines the end type the enzyme will leave, 5', 3', or blunt. The fifth column is the incubation temperature of the enzyme. The sixth and seventh columns provide a general guideline to methylation sensitivity. The eighth column is vendor availability and the ninth column is the cost per unit in '04 dollars.
At the far left, there is a checkbox next to each enzyme name. If you used automatic site selection and are unhappy with one or more of the enzymes chosen, you can check any number of them and hit the "Reconsider" button. Automatic selection will be run again and the enzymes you select will be excluded from consideration.
Your new nucleotide sequence is at the top of this last screen. It would be wise to double check it
in your favorite sequence analysis program to be sure it produces
the correct amino acid sequence at this stage!
You can now take this sequence to another module.