Primer Wizard MANUAL

Primer Wizard MANUAL

- Author: Matteo Ramazzotti
- e-mail: matteo.ramazzotti@unifi.it
- download: Primer Wizard
- source code: code in pdf format.

INTRODUCTION
Primer Wizard software have been developed to speed up the cloning of coding regions of a gene via PCR method. You won't find any "Tell me the Tm and the amplification lenght" or "Find any compatible primer". To clone a coding sequence the user have to respect the start and the stop codons. This is the main rule. All you have to do is to choose compatible Tm for primers and to add cloning tails, if necessary. This is what Primer Wizard exactly do.

INSTALLATION
All you have to do is to unzip all files into the same directory. Three files are required for the program to work:
AA_Triplet: the standard codon usage file.
degen_table: the degeneration table file, according to standard definitions.
modifications: a file containing the list of all available 5'modifications, such as restriction sites.

HOW TO USE
Phase 1: Select Forward and Reverse primers
Once a DNA sequence have been loaded or pasted you can search for start and stop codons into the sequence with the "Find" menu.
The coding frame may be clearer if you see the sequence as triplets. Do it with the "Triplet" menu.
To check if the coding sequence correctly express the protein you're interested in, use the "Translate" menu, by choosing the translation frame.
While the triplet view allows a primer to be selected, the residue view doesn't allow that. The residue view is only a visualization mode.
Once you've find out the start and the stop codons, select with the mouse (i.e. press the left button and keep it pressed while covering the sequence, well, you can also use the keyboard...) a primer containing, for example, the start codon. Mark it by clicking the "Set as Forward primer". The main characteristics of the primer, GC percent, melting temperature and lenght, will be computed and shown into the Forward Primer box, at the upper-right corner (red zone).
Then you have to select a reverse primer containing, for example, the stop codon. Check if the reverse primer characteristics are compatible with the forward one.
If they are not compatible, repeat the primer selection until they are compatible.
Note that in the yellow box, the Reverse primer box, the reverse primer sequence is written in reverse-complementar mode.
Now you can add 5' modification and/or degenerate your primers (or also one primer at a time).
Phase 2-1: Add 5' modifications
Once both primers are decided, click on 5'modifications to set up a for example a restriction site for sticky cloning or to add a regulation sequence upstream the start codon.
If you use a restriction enzyme, check the "Flag" option to add a 5' tail which helps the restriction enzyme to properly and more efficiently cut the DNA.
The Flag can be modified in the Configuration menu, and is intended to be an irrelevant constant for cloning.
Important: the primer characteristics are not recomputed according to the 5'modification and flag, since the annealing is intended to be relevant in the first steps of PCR reactions.
Phase 2-2: Degenerate the primers
By clicking on the "Degeneration" button, a degeneration table will be opened to help you degenerating your primer.
A new window presenting the current primer and its translation will be promped. Change the primer manually for minor degeneration or click on "Auto degenerare" to make a complete degeneration.
The "Test changes" button will translate the degenerate primer, to chack the correctness of the degeneration itself.
The "Done" button will accept the degeneration.
Important: the primer characteristics are not recomputed after the degenaration, and will be expressed as e.g. "About teh old Tm" . Use Tm lower than the original one to ensure an aspecific PCR if you work with degenerated primers!
Phase 3: Accept the primers
You may choose a name for the primer. By clicking on "Accept" button the primer will be fixed and won't be changed unless you "Reject" the primer.
This is a sort of warranty if you work separately on the two primers, to avoid unwanted modifications while working on the other primer.
Phase 4: Generate Report
A complete report af all primer charateristics will be reported in a new window text editor. You can save this report as a .txt file.

MENU DESCRIPTION

The File Menu

Load DNA sequence: opens a sequence.txt file. A filter inside the program will keep only bases (ATCGN) and will discard any other non-DNA character such as line numbers.
Paste sequence: pastes a DNA sequence copied elsewhere. The same filter than in opening procedure is applied.
Close sequence: closes the sequence opend or pasted. All primer choices or informations will be unavailable.

The Configuration menu

Edit modification file: opens the modifications file, to add or remove or modify the 5' modifications.
Be sure to respect the format
Name seque*nce where the * means the cutting site for an enzyme
sequence
to avoid mistakes in modification listing.
Very important: do not use any "-" in modifications, since this is the "end of modifications" mark for the software modification reading algorithm.
Set extremity flag: this is the flag used for restriction enzymes to properly and effectively cut a restriction site added at 5'.
Money matters: How much do I spend for my primers? In report window (see above) this is shown, but the cost per base, the tax nad the money type may be different from user to user.

The Find menu

Start: will mark in the text ATG codon or M (in residue mode). If in not-tripletted mode the frames won't be respected and all ATG will be marked.
Stop: will mark in the text TAA TAG TGA codons or * (in residue mode). If in not-tripletted mode the frames won't be respected and all stop codons will be marked.

The Triplet menu

No explanations. Is clear what the menu opti0ns do. I hope. The Translate menu

Well, I think it's all.
For any comment, suggestion, criticism and so on contact me at the e-mail address indicated above.

Good Job

Matram