Location:
The JCSG Primer Selection Tool is accessible through the main menu by moving your mouse over "Links". A submenu will then appear below "Links." Move your mouse over "JCSG Tools" and another submenu to appear to the right. Click on "Primer Selection Tool" to reach this page.
Introduction:
The Primer Selection Tool allows a user to upload a list of cDNA fragments, define certain values (temperature, salt concentration, etc), and compute a list of primers to split up the DNA. Its output can be seen either in Microsoft Excel, a notepad document whose values are separated by tabs, or as an QIAGEN Operon Ordering Form.
More information about Primer Selection Methodology can be found here.
Below is a picture of the Primer Selection Tool. Click on the area of uncertainty for an explanation.
Steps to Get Primers:
Optional input. The only required file is the Sequence FASTA file (see Step 1b) In addition to a sequence file, a tab-delimited file can be uploaded by the user. That file should contain the accession code of the target sequence, found in the sequence file, followed by the start and end coordinates of the subsequence to be amplified.
This capability is especially useful to generate multiple primers from a single cDNA without having to prepare multiple subsequences in FASTA format, and also to reprocess a target list with optimized constructs/truncations.
Here is an example of using the Target List upload to process different start/end positions on the same target:
|
Gene Name |
Start |
End |
|
fasta_target1 |
1 |
247 |
|
fasta_target1 |
1 |
104 |
|
fasta_target1 |
110 |
200 |
NOTE: Make sure the Gene Name corresponds with the same name found in your FASTA formatted file.
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1b. Sequence FASTA file (required)
You must provide the FASTA in order to run the Primer Selection Tool. The input to the public primer generator is an uploaded flat text ASCII file containing single or multiple nucleic acid sequences in FastA format.
The first character of the first line is expected to be a ">". It is immediately followed by the sequence identifier, a single word, which is used to identify sequences in the primer generator output. Further words on the same line are considered as comments. The sequence begins on the next line, and follows until the end of file is reached or another line is found which begins with a ">". White spaces, tabulations and newline characters can be inserted within the sequence, and will be ignored. In the public interface, sequences may contain or exclude stop codons. There is no limit to the number of sequences that can be uploaded or the sequence length in both public and internal interfaces. Sequence data is case insensitive.
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Type in the operation temperature at which you wish to use the cDNA fragments at. The default temperature is 62.5 degrees Celsius.
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You can also specify the Tm tolerance, which corresponds to the acceptable range of temperature over and below the optimal Tm for which primers are acceptable. Default for the temperature tolerance is +/- 2.5 oC.
For example, at a temperature of 62.5 degrees, the temperature will range from 60 to 65 degrees, thus allowing more primers that fit into the five degree range.
NOTE: If you change the tolerance to 0, the program may not give you any possible primers since none might fit exactly at the specified temperature. For example, for a temperature selection of 60 degrees Celsius and a Tolerance of 0, primers working at 60.1 or 59.8 will result in empty findings.
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Type in the amount of salt present in the experiment, with the default being 100 uM.
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The Tm option allows the user to select the optimal melting temperature (Tm) in degrees Celsius for the primers designed. Tm calculations in the JCSG Primer Generation Tool are performed according to the equation used by QIAGEN Operon, based on the Meinkoth & Wahl Long Probe method. The formula used by the JCSG Primer Generator Tool is:
Tm = 81.5 + 16.6 log [Na+] + 41(#G + #C)/length - 500/length
where [Na+] is the sodium concentration, #G and #C are the number of Gs and Cs bases in the nucleic acid sequence, and length is the length of the sequence. This formula is recommended for oligonucleotide lengths ranging from 20 to 100 residues.
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Sequence length constraints: Specificity, Tm, and time of annealing are at least partly dependent on primer length, making this parameter critical for successful PCR. In general, oligonucleotides with lengths between 18 and 24 bases are very sequence
specific.
Longer primers will give higher specificity, but oligonucleotide cost increases with length. Many suppliers have 35 bases as an upper limit to switch from 10 nmol to 50 nmol scale synthesis, substantially increasing primer cost. Therefore, the default
minimal and maximal lengths for primers generated through the JCSG Primer Generation Tool are set at 18 and 35 bases, respectively.
NOTE: Be sure to look at the Theoretical bp Range to see what the calculated lengths are. Any minimum sequence higher than the second value in the Theoretical bp Range will always turn up an empty search since such a high value is not possible.
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This is the only white field that you can not type into directly. The theoretical bp range is automatically calculated based on user inputs for temperature, tolerance, and salt concentration.
Use this range as an advice for selecting minimum and maximum primer sequence lengths.
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8. Forward/Reverse Primer Restriction Site
Forward Primer Restriction Site -
There is no default restriction site for the forward primer.
Reverse Primer Restriction Site -
The reverse primer has as a default the following sequence: CCCGGCCGGCCCTA, which corresponds to a restriction site for the enzyme FseI.
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The Prepend ATG option is useful for segments where a methionine is not at the beginning of the sequence. The default is checked. If a sequence starts with a methionine codon (ATG) this option is ignored.
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10. Convert xTG to ATG on full or truncated sequences
Convert xTG to ATG option on full or truncated sequences is applied to some bacterial sequences where the start codon is listed as XTG. The default is checked. If the start codon is ATG, this conversion is ignored.
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In the Output File Format option, you can choose between three file formats offered: a Microsoft Excel file, a tab delimited text file, or a QIAGEN Operon Ordering Form. The default is a Microsoft Excel spreadsheet. The Primer Generation Tool writes the oligonucleotide primer sequences in 5'3' orientation.
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After completing the previous eight steps, click the button to get the calculations on the specified file (containing data in the proper format) with user-specified values.
NOTE: The creation of a primer list takes about a minute (depending on the size of your list), so please be patient.
Following the input of the sequence data, the program would strip off the stop codon, if that option was checked. Next, the FastA description header information would be processed and the first word would be used as the sequence identifier for the output. Subsequently, a collection of primers with Tm's within the ranges specified by the user is calculated for each target sequence. If the Tm's for primers within the length constraints defined by the user do not fall within the Tm and tolerance limits, the primers are discarded.
Because of the speed of the program, minor changes in Tm tolerances and primer length can be introduced and the program can then be rerun. In most cases, those minor changes would be enough to force the identification of acceptable primers. It is well known that the 3'-terminal position in PCR primers is essential for the control of mispriming. The presence of G or C bases at the 3' end of primers (GC clamp) helps to promote correct binding at the 3' end due to the stronger hydrogen bonding of G and C bases. GC clamp scores are calculated based on the 3 last bases of each primer according to the following schema: [GC][GC][GC] = 0; [ATGC][ATGC][AT] = 1; [ATGC][AT][GC] = 2; and [AT][GC][GC] = 3, with 0 corresponding to the worst GC clamp and 3 corresponding to the best, respectively.The program selects the best forward and reverse primers and highlights them with asterisks in the output file.
The chosen primer for each target corresponds to the primer within the selected Tm range that has the highest GC clamp score. Finally, the program generates the output according to the user specifications. Independently of the output format options selected by the user, the program outputs a summary page with the parameters used for the calculations and well as the number of successes and failures. A pop-up window contains the output files, including raw data, the user-selected output files, exception/failure logs, etc. Separate forms are produced for forward and reverse primers. If a primer pair failed, it is flagged as failed and the causes for the failure are reported in the exception/failure logs. The program not only calculates and outputs the Tm's for the selected primers, but also their sequences, lengths, GC clamp scores, and GC content in percentage value. Ideally, the base composition of primers should be between 45% and 55% GC, although we have not experienced PCR failures due to lower GC contents as long as the primers have optimal 3'-terminal GC clamps. Therefore, acceptance or rejection of primers based on their GC content is left to the operator. In case the primer design process fails under the user-selected conditions, users can re-evaluate the input parameters and perform another full calculation to maximize the number of successful primers. Alternatively, users can extract the sequences of the failed targets listed in the exception/failure log and perform a second cycle of selection under slightly different conditions restricted to the failed sequences.
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Click the button only if you messed up and would like to clear all the filled boxes or if you have another file you would like to test for primers.
Click here to return to the Primer Selection Tool picture.