SGD Help: Primer Design

This is the help document for SGD's Primer Design tool, which facilitates the design of primers that will be located outside of a region of DNA that you define (sourced from either a selected yeast gene or from pasted-in DNA sequence); the tool is based on the Primer3 program, utilizing the Primer3-py package.

List of steps (see below for details on each)

1. Enter DNA source to be used for searching for primers
2. Input start and end positions of your desired target region
3. (optional) Input a maximum product size
4. (optional) Force endpoints of primers
5. Primer picking parameters
6. Results table
7. Troubleshooting
8. More about the Primer3 package
9. Go to Primer Design

Using SGD's Primer Design

(based on Primer3 program; see below for more information).

Step 1. Enter a DNA source to be used for searching for primers (uppermost input box; required field). Choose only one of the following options:

• A. Enter a gene or ORF name that is present in the S288C reference genome.

OR:

• B. Paste in your own DNA sequence. Enter ONLY DNA sequences, without any headers, comments, spaces or numbers, etc. Carriage returns are OK if they do not precede the sequence. A good example is a "fasta" format sequence file with the header line deleted. Generally the sequence should be ~200 bases or longer.

Step 2. Input “start” and “end” positions of “Target Region” (required fields). Note that 500 and 900 are shown on the default screen as example input values; change them to alter the region for which you want to design flanking primers.

• A. "Target Region" for Gene/ORF name entry:

If you entered a gene/ORF name, the start and end positions of your "Target Region" are relative to the ATG start codon of the gene’s coding sequence, where the A of the ATG is position "1". We use the SGD reference genome (S288C) sequence for the gene as the basis to pick primers. Our program can search the regions 1000 bp upstream and downstream of the gene's coding sequence; note that negative numbers indicate positions upstream of the ATG (for the purposes of this program the nucleotide immediately upstream of the A of the ATG is considered as position "0", going to -999 bp upstream of the ATG). Positive numbers continue through the genomic coding sequence of the gene (including introns if present) and can continue past the stop codon to 1000 bp past the stop codon. IMPORTANT: Note that the program will look for the best primer pairs flanking your target region, so it will only look OUTSIDE of the start and end positions you entered; it will try to find the best pairs as close to your target region as possible, but it needs a reasonable amount of sequence in which to search. To obtain primers that flank the start and stop codons of the entire gene’s coding sequence, increase the 'End at the 5' location from START codon' to the whole ORF length to be the length of the gene (found from Sequence information on the gene’s Locus page) or increase the maximum product size to a size larger than the gene’s size.

• B. "Target Region" for pasted-in DNA sequence:

If you pasted in your own DNA sequence, enter the starting position and the ending position of the “target region” that you are interested in, relative to the first base of the pasted in sequence. Exactly as for the IMPORTANT note above, the program will only look OUTSIDE of the start and end positions you entered as inputs. If you do not get results, it could be because there is not enough room in the flanking regions to find primers and you may need to make your “target region” size smaller, or paste in new DNA sequence with more flanking region sequences.

Optional Step 3. "Maximum Product Size".

To customize the size of your desired product, and/or if you would like to make sure that the primer pairs fall within a certain maximum size range, you can enter a value here that must be larger than the “Target Region" size (the end position minus the start position of your input values). As discussed above, the program will only look OUTSIDE of the target region start and end positions you entered and will try to find the best pairs as close to your target region as possible, but it sometimes needs a reasonable amount of sequence to search. If you do not get any results with your Maximum Product Size, it could be because there is not enough room in the flanking regions to find primers, and you may need to make the Maximum Product Size larger, or make the target region size smaller (or paste in new DNA sequence with more flanking region, if you pasted in DNA). Note that the program will try to come close to the Maximum Product Size, so you can customize your desired product size and region by using this option in conjunction with your target region.

Optional Step 4. Force start positions of primers to be at the start and end positions of the “Target Region".

Setting this button to “Yes” will force the 5’ base of the forward primer and the 5’ base of the reverse primer to be exactly the position entered as the “start” and “end” position, respectively, for the "Target Region" as described in Section 2 above. IMPORTANT: Note that the default primer picking parameters are fairly stringent and that in many cases you will not get any results when trying to force the ends of the primers to certain positions. You may have to relax many of the parameters or try slightly different positions for the primers.

Step 5. Primer picking parameters.

You can change parameters for primer length, GC% composition, melting temperature and various complementarity/annealing allowances. The defaults used for our program are the same as those used for the Primer3 program at the http://primer3.ut.ee website ("download settings" button). You can click on the name of each parameter to get linked to a definition at the Primer3 website. See below for more information on Primer3.

Information about primer parameters:

• Primer composition - Primers that contain a skewed AT/GC ratio can fail to give high specificity, or be poorly behaved in other ways. You can enter minimum, optimal, and maximum values for the percentage of basepairs which are either G or C.
• Primer melting temperature - Melting temperature heavily influences the results of PCR. Minimum, optimum, and maximum values can be set by the user.
• Primer Annealing - Primers also tend to dimerize and anneal to themselves, which can present significant problems in using PCR. For a visual representation of this problem, see http://bioinfo.ut.ee/primer3-0.4.0/primer3/input-help-040.htm ; maximum allowable alignment scores for annealing between primers can be set by the user.

Step 6. Results table.

If the program finds primer pairs for you, the “best” pair of primers (based on best match to your chosen parameters and the least amount of annealing/complementarity) will be shown at the top of the results table with the suffix number of “0”, then the second best pair (suffix number “1”), and so on. There will be a maximum of 5 primer pairs shown, but sometimes fewer will be shown if there were no other pairs that pass the minimum allowable parameters. If no primer pairs are shown, you need to adjust parameters and/or target size and/or amount of DNA pasted in, as described above. Note that if you chose to enter a gene/ORF name as your search input, the “start” position of the primers are shown relative to the ATG start codon (and can be negative if primers are upstream of ATG). However if you pasted in DNA as search input, the “start” position is relative to the first base in the pasted in DNA sequence. The following columns in the results table are: “product size” = size of amplified product (including primers) for a given primer-pair sharing the same number suffix; “tm” = melting temperature; "gc%" = GC percent; "any_th", "end_th", and "hairpin" are complementarity/annealing scores (see below), and “seq” = the sequences of the primers, shown 5’ - 3’.

Definitions of the complementarity/annealing parameters shown in the results table; the lower the number, the better (note the Primer3 defaults are fairly stringent and thus any primers that are generated should generally be considered "well-behaved"):

• “any_th” (Self Complementarity) see: http://primer3.ut.ee/cgi-bin/primer3/primer3web_help.cgi#p3w_primer_any = the self-complementarity score of the primer (taken as a measure of its tendency to anneal to itself or form secondary structure).
• “3’_th” (Self Complementarity) see: http://primer3.ut.ee/cgi-bin/primer3/primer3web_help.cgi#p3w_primer_three = the 3' self-complementarity of the primer (taken as a measure of its tendency to form a primer-dimer with itself).
• “hairpin” (Primer or Oligo Hairpin) see: http://primer3.ut.ee/cgi-bin/primer3/primer3web_help.cgi#p3w_primer_hairpin = the calculated value of the melting temperature of the most stable hairpin structure of the primer.

Troubleshooting:

If the program does not show any primers in your results table, then it was unable to find primers using the parameters and sequence regions you inputted. You can try several approaches to have a better chance of generating primers. The default primer picking parameters are fairly stringent so you can try relaxing the parameters (e.g., allow a wider range of GC% or larger ranges of melting temperature or primer lengths). As noted above in section 4, when trying to force the ends of the primers to be at certain positions, in many cases you will not get any results; you can try inputting slightly different positions for the primers if possible. Finally, there may not be enough sequence to search in the regions flanking the target region defined by your input start and end values. You may need to make the the target region size smaller (or paste in new DNA sequence with more flanking region, if you pasted in DNA), or make the “maximum product size” larger.

More about the Primer3 program:

SGD's Primer Design tool uses the Primer3-py python package: https://pypi.org/project/primer3-py/0.5.5/

Primer3 source code available at https://github.com/primer3-org/primer3

Web-based Primer3: http://primer3.ut.ee

Help pages for Primer3:

• http://primer3.ut.ee/cgi-bin/primer3/primer3web_help.cgi
• http://bioinfo.ut.ee/primer3-0.4.0/primer3/input-help-040.htm

Citations:

• Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M and Rozen SG. Primer3--new capabilities and interfaces. Nucleic Acids Res. 2012 Aug 1;40(15):e115.
• Koressaar T and Remm M. Enhancements and modifications of primer design program Primer3. Bioinformatics 2007;23(10):1289-91.

Go to SGD's Primer Design