This tool was developed to meet the demands of primer design for highly variable sets of aligned sequences. It includes design requirements for next-generation sequences (NGS) including 454 sequences. It can also be used for primer and probe design for PCR, Sanger sequencing, and other systems with custom barcodes and DNA handles for universal primer annealing. The tool will design several alternative primer sets, whenever possible.
Please cite these papers when using PrimerDesign-M:
If you have any difficulty, or want to suggest improvements, contact us: firstname.lastname@example.org.
Premade sequence alignment input
This option provides several choices for inserting a premade alignment for HIV-1, HIV-2, or SIV. The following alignments are offered:
For additional details about these types of alignments, see our Alignments page.
Gap Stripping: This option will remove all columns of the alignment having more than the selected percentage of gaps.
Region of interest: This is the region you need to amplify from your alignment, i.e., the minimum part that you absolutely need amplified. Your alignment must include additional sequence on either side of this region; MORE sequence is better (within reason, and within the length restrictions of the sequencing and PCR methods you are using). The Start and Stop coordinates of the Region of Interest are the nucleotide numbers of your input alignment, not reference sequence coordinates.
Multiple fragments: Select "single fragment" if you are seeking options for making ONE pair of primers. Select "multiple fragments" if you want to make a SET of primer pairs spanning a longer region of interest. See Output examples below.
Fragment overlap: When finding multiple primer pairs, these options dictate how much overlap will exist between each successive primer pair.
- Max tries to position the next forward primer in the first 25% of the fragment; if unable, the next quarter is considered, and so on.
- Mid tries to position the next forward primer near the middle of the fragment; if unable, regions on either side of the midpoint are considered.
- Min tries to position the next forward primer in the last 25% of the fragment; if unable, then the previous quarter is considered, and so on.
- Zero uses the reverse primer from one fragment as the forward primer of the next fragment.
- Flex takes a flexible approach in choosing the position of the next forward primer.
No adaptor: No DNA handle will be attached to the construct.
Read Length: If you choose "No adaptor", you need to provide a read length. This length depends on the read length of the system you are designing primers for; different sequencing technologies have different read length limitations. The read length should be the expected read length minus one primer length. For "single fragment" primer design, the read length must be longer than the Region of Interest (after gapstripping).
If you choose "Multiple fragments", you will need to choose Min and Max read lengths. These lengths will dictate the length of your fragments.
GS FLX adaptor: These 454 adaptors will be automatically attached.
Adaptor A: GCC TCC CTC GCG CCA TCA G Adaptor B: GCC TTG CCA GCC CGC TCA G
GS FLX Titanium adaptor: These 454 adaptors will be automatically attached.
Adaptor A: CGT ATC GCC TCC CTC GCG CCA TCA G Adaptor B: CTA TGC GCC TTG CCA GCC CGC TCA G
My adaptor: Specify your own DNA handle sequences. When selected, 2 input windows will appear for you to paste your adaptor sequences.
You can choose how to optimize the tags/barcodes. In each case, one of the 3 optimization parameters is optimized, while the other 2 are specified by user input. For a more detailed explanation of these constraints, see Figure 3 of Brodin et al. 2013.
No tag: No tags will be attached to the construct.
Optimize number of tags: Get as many tags as possible, given the user-specified length and edit distance.
Optimize length of tags: Get the length to be as short as possible, given the user-specified number and edit distance.
Optimize edit distance: Get the edit distance to be as long as possible, given the user-specified number and length of tags. This can be used to ensure the tags are at least a certain number of mutations apart.
Minimum primer length: This is the minimum length the primer part of your construct, i.e., the minimum length you want the organism-specific detecting oligomer to be. The accepted range is 10 - 40 (10 ≤ Length ≤ 40 nucleotides).
Maximum primer length: As minimum length, but maximum length you want the organism-specific detecting oligomer to be. The accepted range is 10 - 40 (10 ≤ Length ≤ 40 nucleotides).
Detection Limit (%): This determines the level at which rare variants should be included in the design. If included, then primers may contain degenerate positions (IUPAC codes) that anneal to sequences found in the alignment down to that level.
Complexity Limit: Complexity, in this context, refers to the number of possibilities incurred by having IUPAC codes included in the primer sequences. For example, the sequence GTATGGGCAAGCAGGGARMTGG contains one R (A or G) and one M (A or C), which yields a total of 4 possible sequences for this primer (complexity = 4). Choosing a low Complexity Limit will constrain your primer choices to avoid regions with degeneracy, but it may result in failure to find primers (depending on the stingency of other constraints).
Max Difference in T
Dimer Window Size: The size of the window in which dimerization is investigated. This size window moves across all potential primer-primer interactions.
The default Dimer Window Size (10) is based on published experimental results in Desmarais et al. 2012, Electrophoresis 33: 483-491. PMID 22331820.
Dimer Max Ratio: The maximum ratio of matched nucleotides within a dimer window. If a potential dimer is detected at or above this ratio, then that set is discarded.
The default Dimer Max Ratio (0.9) is based on published experimental results in Desmarais et al. 2012, Electrophoresis 33: 483-491. PMID 22331820.
G/C Clamp: The G/C clamp options allow you to specify that the 3' end of the primer must be a G or C. For multiple fragments with option Fragment Overlap = Zero, the G/C clamp will, of necessity, be on both the 3' and the 5' ends of each primer. For other input (single fragment or multiple overlapping fragments), the G or C will be only on the 3' end.
Email return will occur automatically when a computationally demanding design is requested. You may also choose "Always email results", which is recommended if your internet connection is slow or if you experience a browser timeout.
Below are 2 examples of tool output.
Example of output for a single fragment:
Example of output for multiple fragments:
Return to PrimerDesign-M tool interface.