ORFprimer - free PCR primer design software

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ORFprimer manual Search sequences Calculate primers Save primers and sequences to local database Set Preferences Input File Format Additional Tips & Tricks Search sequences Search GI or accession Enter a GI identifier (a number) or an accession number. Numbers will be interpreted as GI identifiers and entries containing letters will be interpreted as text. Open search list Prepare a text file with one GI identifier or accession number per line. From the "Open Search List" menu item select the file you have prepared. ORFprimer will start searching for the sequences in the file immediately. You can also note positions of the PCR primers in the same file. See below for details. Get all Sequences in local database As it is described in the save menu you can save the sequences that you downloaded from the NCBI webserver to a local database. In some situations one needs to obtain these sequences to e.g. create a new pair of primers. Therfore this option provides the possibility to fetch all sequences from your database. Clear search list ORFprimer won't find sequences if the GI identifier or the accession number of the protein or dna sequence is not correct. These sequence are displayed in the tables with the grade "search failed". This is an important feature because the user wants to know which sequences were found and which were not. Anyway, if the correct sequence was found by specifying the correct identifier, the user may delete all sequences that were not found with this option. Repeat Search You can start the search for all sequences that are displayed on the first two tabs once again. Abort Search A search for more than thousand sequences at once will take some time. Especially if the data files downloaded from the web server at NCBI are huge or your internet connection is slow, you will have to wait some time. You can stop this procedure and abort the search at any time of this progress. Reset If you have finished a computation of primers for a set of sequences or if you searched for the wrong set, you can clear the list with this item. Exit program You leave the ORFprimer program by clicking on this menu item or by clicking on the cross in the right upper corner or by pushing `Alt + F4` Calculate primers for a Sequence from fasta file You can calculate primers for a sequence already downloaded to a file in fasta format. If you use this option a new window will open, enabling you to browse through the diectories on your computer and to choose the fasta file. Since ORFprimer is a software designed to compute primers in a high throughput manner we do not recommend to use this but one of the following items. for all rows on 'results' tab The second tab titled "Search Results" lists all Sequences obtained by ORFprimer. You can calculate PCR primers for every sequence with this option. As a default ORFprimer will set the upper primer at the beginning of the open reading frame and the lower primer at its end. for selected rows on 'results' tab On the "Search Results" tab you can specify the sequences you intend to compute primers for. ORFprimer will calculate primers for the beginning and the end of the sequence. If you want to compute a single primer for a sequence or if you want to add/delete primers from the third "Primer overview" tab have a look to the Tips & Tricks section. use aa positions This box is checked as default. That means that all positions which are shown on the third and fourth tab indicating the primers positions relative to the corresponding sequence point to amino acid positions. We recommend to use this option to avoid confusion but its also possible to uncheck this box: all positions will be displayed pointing to regions of the dna sequence. Save primers and sequences to local database Save fasta protein ORFprimer will save all protein sequences to one Fasta file, if you choose "Save Fasta Protein" in this menu. If you want to save only a set of sequences, check the boxes in the "Selected" column in the "Search Results" table of those sequences and select "Save selected only" in this menu. Save fasta DNA ORFprimer will save all DNA sequences to one Fasta file, if you choose "Save Fasta DNA" in this menu. If you want to save only a set of sequences, check the boxes in the "Selected" column in the "Search Results" table of those sequences and select "Save selected only" in this menu. Save to local database Set up the local database as described. If you choose not to save the database password, make sure to enter it again upon program start. By default, all sequences are saved. If you want to save only a set of sequences, check the boxes in the "Selected" column in the "Search Results" table of those sequences and select "Save selected only" in the "Save" menu. Save primers to local database Set up the local database as described. If you choose not to save the database password, make sure to enter it again upon program start. By default, all sequences are saved. It is not possible to choose subset of primers to be saved to the database. As it is described in the Tips & Tricks section you can easily add or delete primers. Therfore all primers being calculated should be correct. Furthermore ORFprimer can only write to your database, but is not intended to work as a database client to manage your data. Consequently you should check if all the primers are correct before writing them into your database. If a primer sequence is already present in your database ORFprimer won't add it another time with a different name. Save tab delimited This format is suited for opening with Microsoft Excel. In this menu choose "Save Tab delimited". By default, all sequences are saved. If you want to save only a set of sequences, check the boxes in the "Selected" column in the "Search Results" table of those sequences and select "Save selected only" in this menu. Save selected only This box should be checked if you want to save only a subset of DNA or protein sequences to your database. Since only DNA and protein sequences displayed in the tables of ORFprimer provides the option to be selected, this obviously does not work with primer sequences. Set preferences If you use Java Web Start, preferences will be stored somewhere in the Java Web Start directory. Otherwise, a file orfer.properties will be generated in the directory that ORFprimer was started in.The Preferences can be used to adjust the way ORFer does the searching. The Preferences are also used to set the local database connection. "Search options" tab NCBI XML sequence files often contain more than one sequence entry. Choose "Retrieve only exact match", if you want the exact sequence that was searched only, otherwise choose "Retrieve all sequences in XML files". If there are sequences stored in the local database, choose "Search in local database first". ORFprimer will try to get the sequences from the local database, which saves time and bandwidth. "Use local XML files": XML files retrieved from NCBI can be stored in a local folder and reused later. Saves time and bandwidth. "Calculation options" tab The first setting you can specify on this tab affects the design of the primers sequences: it is possible to add a spacer to the left (upper) and right (lower) primers sequence. This means that the sequence that you type in to the two textfields will be inserted between the recognition site of the enzyme and the complementary dna sequences. For example if you choose BamHI (recognition site: `GGATCC`) to be the enzyme for the upper primer and you specify `AAAAA` as the spacer sequence, the resulting primer could look like this: `ACGGATCCAAAAACOMPLEMENTARYDNASEQUENCE`, where the first `CA` is a sequence that improves the recognition of BamHI (for other enzymes this sequence will differ), and `COMPLEMENTARYDNASEQUENCE` is the primer sequence complementary to the DNA sequence. The following four options correspond to the PCR settings you use: you should specify the DNA concentration, monovalent salt concentration, magnesium concentration, and the DNTPs concentration. If you don't change these settings, the primers being calculated could nonetheless be correct. Anyway, the computed melting temperature is more precise if these values are exact. Finally, you might define the minimum and maximum length and the melting temperature you preferably use for your experiments on this tab. Clearly, the value for the maximum length should not be lower than the value for the minimum length, because this does not make much sence. The same holds for the desired melting temperature: do not specify a value above 90 or below 40 degrees Celcius. "Enzyme options" tab This tab provides options to change the recognition sites for enzymes inserted into the primers sequences. You can see two pull down menus at the upper side of this tab. Here you should indicate which enzymes you use to cut the amplified DNA sequneces for the left (upper) and right (lower) primer. ORFprimer will insert the recognition sites of these enzymes into the primers sequences if they are not present in the DNA sequence multiple times. In that case the cutting of the DNA would occur at all these positions and therefore the resulting DNA product could not be correct. To avoid such behaviour ORFprimer autmatically uses alternative enzymes which can be specified in the two lists below. These list display all enzymes you can alternatively use in alphabetical order. All selected enzymes are shown on top of a alphabetical sorted list with a number in the left column. This number indicates the order which is used by ORFprimer to insert these sequences into the primers sequence. For example, if the first to enzymes fail to be inserted (because the recognition sequence is present one or more times in the DNA sequence), the third enzyme sequence would be chosen. You can change this order by clicking on the cells in the left colum of the lists. This will open a pull-down menu. It is recommended to select some enzymes that could be alternatively used. ORFprimer will automatically try to use the best one. You can change the enzymes after the calculation of primers is already finished. "Edit/change enzymes" tab On this tab all enzymes are shown in the list on the left side. You can mark one of these enzymes and change it by clicking on the "change enzyme" button. This is an advanced option. A window will open which makes it possible to change the tail of the enzyme. In the exampe above ("Calculation options" tab) we explained that the sequence `CA` improves the recognition of an enzyme to its site. You can change the length of this tail if you experiences that the results are better using a different length. ORFprimer will optimize this sequence to avoid the formation of loop structures. If you can't find the enzyme you would like to use for your experiments, you can add the enzyme by clicking on the "create enzyme" button. Input File Format Input file for automated primer design Sequence searching and primer design can be done manually, or by preparing a text file with a list of primer positions. The text file can have two formats. Either, a simple list of GI or accession numbers, or a list with GI/accession numbers, position and strand (upper/lower). Example Tips & Tricks Sort data Click on the header of column to sort the data in alphabetical ascending order. Click again to sort in descending order. Copy & paste from ORFprimer to other applications You can copy data from the "Search Results" or "Search List" tables and paste the data to other applications. Use the right mouse button to open the copy/select pop-up menu. (Thanks to the SQuirrel-SQL project for the code of the pop-up menu). You can paste multiple (or all) fields in the table to Excel. With Linux, use xclipboard to get hold of the data that was copied. Add / delete primers In most cases one wishes to compute two primers corresponding to one specific sequence: one upper primer at the beginning of the sequence and a lower primer at the end. But this rule doesn't hold for every situation. Sometimes it might be necessary to compute more than two primers for one sequence or only a single one. Therefore we used a modification of the pop-up menu described above. This pop-up menu is exclusively available on the third tab titled "Primer overview". To delete a specific primer you should mark this primer via left-click and open the menu via right-click with your mouse. Similarly you can add a primer to a marked sequence. How to set up your own database To store sequences in a local database, prepare a MySQL, MS Access or Oracle Database and enter your database parameters in the ORFprimer preferences. Use the drop down menu "Select URL template for ..." to quickly generate a URL string for your type of database. Saving the password is not recommended in the current version, because the password will be saved without encryption in the orfer.properties file. First of all you'll have to make the database: MySQL Download the orfer_mysql.sql SQL Script. Run the script: `mysql < orfer_mysql.sql` to create the necessary objects in an MySQL database. Enter "jdbc:mysql://[IP or hostname of your database server]/orfer" as URL in the ORFprimer preferences. Enter MySQL User name and password in the preferences. Access Download the Access database orfer.mdb (Microsoft Access 97 format) Create ODBC System DSN named `orfer` with orfer.mdb as the data source. This is done with the Data Sources (ODBC) manager in the control panel. Enter "jdbc:odbc:orfer" in the ORFprimer Preferences. Oracle Download the orfer_oracle.sql SQL Script to create the necessary objects in an Oracle database. Run the script in SQL-Plus Enter "jdbc:oracle:thin:@[IP or hostname of your database server]:1521:[SID of the database]" as URL in the ORFprimer preferences. Replace IP, SID and Port (default 1521). Enter your Oracle user name and password in the preferences. Non-interactive Mode ORFprimer can be run in non-interactive mode by excuting ORFprimer.jar from the command line with a the file name of list of GI identifier or accession numbers as argument. ORFprimer.jar is included in the file orfprimer-x.x-bin.zip, which can be downloaded at SourceForge `java -jar ORFprimer.jar gi_liste.txt` All protein and DNA sequences will be written to the files `pep.fasta` and `dna.fasta` in the local directory, respectively. Place a file `orfer.properties` (example file) with the details of the database connection in the same directory. All retrieved sequences are automatically stored in the local database. Search failed Search might fail for certain sequences. Some of the XML files sent by the NCBI are malformed and cannot be parsed by ORFprimer. The reason could also be an interrupted internet connection or a high load on the NCBI web server. If an accession number was used to search, the search should be repeated with the respective GI identifier. It might be helpful to display general output and error messages on a Java console window. To display the Java console, set the appropriate options in the Java Web Start Application Manager in Files/Preferences/Advanced.