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Codon Optimizer

A software application that optimizes the codon distribution in sequences

The user specifies a sequence composed of characters in the set {A,C,G,T}, corresponding to the base-pairs Adenosine Cytidine, Guanosine, Thymidine, in the textbox in the Codon Optimization window, choses a codon usage source such as "Eukariotic", may select one or more restriction enzymes to prefix or append on the 5' or 3' ends of the strand, and press the Submit button to display the result in the Optimized Codon window, in which an "optimized" sequence is displayed, along with a primer sequence.

Since there are 20 amino-acids found in nature, and each can be represented in triplets (codons) formed using four alphabetical characters (nucleotide base-pairs), of which there are 64 distinct permutations, then an amino-acid could be represented by one or more triplet (the inverse is not true:  a specific triplet cannot represent wo or more distinct amino-acids).  For example, Alamine can be represented by four codons:  GCA, GCC, GCG, GCT.  However, depending on the organism, the probabilities of finding a specific codon for a given amino-acid varies.  Using the Alamine example, in eukariotic organisms, the frequence of finding the GCG codon is 17%, while in bacteria (E.Coli) is 36%, and in yeast it's 11%.

The application takes the sequence as specified by the user, divides it into triplets, and for each triplet (codon), it determines its associated amino-acid, generates a number between 0 and 100, looks up the probability distribution of the amino-acid's codons, and picks he one that best suits the random number.  The result is an "optimized" sequence that more closely matches one found in nature for the specified organism (codon source usage).

The application also prefixes at the head and appends at the tail of the optimized sequence up to four restriction enzymes.  The restriction enzymes are selected by the user by clicking on the checkbox below a listbox, which enables the listbox and allows the user to select an enzyme.  Selecting an enzyme also enables the left-adjacent checkbox, thus allowing the process of selecting an enzyme to flow from right to left, from position 1 to position 4.  This flow controls the selection of enzymes to produce an ordered list of enzymes.

Restriction enzymes can be added and removed from the list by using an Enzyme Editor that can be opened as a dialog.  In it, the user specifies the name fo the enzyme as it would appear in the listbox, and its code sequence.  The code sequence can be composed of "wild" characters, {R,Y,N,B,D,H,V,K,M,S,W}, which can respectively be any character in the groups [AC],[CT],[ACGT],[CGT],[AGT],[ACT],[ACG],[GT],[AC],[GC],[AT].

The application uses pre-defined codon distributions for Eukariotic, Bacteria, and Yeast codon sources, as determined by recent research publications.  The user is allowed to define custom sources, and specify its codon probability distribution values.  The data entered is then automatically saved to a file by the application, and read when the application is launched later.  The added new codon source will appear on the listbox in the Codon Optimization window.

The user can also specify micro-sequences within the original sequence that should remain fixed and not allowed to be replaced by the optimizer.  Using the Fixed Sequences dialog, the user types and adds to a list values such as TGG or CCGG, and if such sequences are found in the original sequence, the application guarantees that they will appear in the same position, unchanged, in the resulting optimized sequenced.

On similar style, the user can also specify micro-sequences that if found within the optimized sequences, the application must re-submit the original sequence for another optimization until such "excluded sequences" are absent.  If after 1000 attempts in optimizing, no resulting optimized sequence is produced that fullfills its validation requirements, no resulting sequence is produced and an informative message is displayed to the user.

A special case applies when selecting a NcoI restriction enzyme on the 1st position on the 5' end of the strand, and the original sequence begins with "atgg".  Since the NcoI enzyme is defined to have a code sequence of "ccatgg", the application would normally prefix the sequence with the the enzyme's code, resulting, in this case, with "ccatggatgg...".  In this particular case, only the code "cc" is added as a prefix.  Furthermore, if the 4th character of the original sequence is not a "g", the user is prompted for wether he/she wishes to have the application replace it with a "g" automatically.  Choosing not to will not consider of applying the NcoI at all.  Similarly, checking the Kozak Sequence checkbox does the same thing, except that in this case it uses a different code sequence to prefix the optimized sequence.

Selecting (and highlighting) text in the sequence textbox, displays the selected text's starting and ending position within the sequence, along with its offset of 3, and its length.  Simply positioning the insertion cursor within the text, displays its position an offset.  Selecting the Find menu item in the Edit menu opens a Search dialog in which text can be specified and used to search such text in the sequence, and if found, the found text in the sequence textbox is highlighted with the Search dialog still open.  Pressing again the Search button in the dialog finds the next occurrence in the sequence.

The Primer Prefix textfield is used to specify a text used to prefix every row in the primer text.

The Protein checkbox is used to inform the application tha the sequence specified in the textbox is not describing a sequence of base-pairs, but amino-acids, using the one-character symbols for each of the 20 amino-acids.

In the Optimized Codon result window, the user can select from three possible viewing options:  ORF (open reading frame) only, ORF + restriction enzymes, ORF + enzymes + stuffer.  The stuffer is a sequence of randomly-generated characters whose length is such to allow the primer to be formatted in a certain size and shape.  Care has been taken to ensure that the stuffer doesn't contain any of the STOP codons, or the ATG codon.

For storage, the resulting optimized and primer text can be saved to a text file.  The optimized codon, along with all its attached values such as list of restriction enzymes, codon source, kozak sequence usage, can also be saved to a proprietary-formated data (binary) file, which can be used only with this application.  This latter file can be used to launch the application when double-clicking it in a Windows operating system and its CDN file extension associated to the executable file the application, CODONOPT.EXE, or it can be read in into the application by selecting such operation from a menu item choice.

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