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Primer Design Explanation

Background

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 this paper when using Primer Design:

If you have any difficult, or want to suggest improvements, contact us: seq-info@lanl.gov.

Input

Requirements:

Primer Options

Region of interest: Enter the Start and Stop coordinates of the region you need to amplify from your uploaded alignment. This is the minimum part that you absolutely need amplified. Your alignment must include sequence on either side of this region, the more the better (within reason, and within the length restrictions of the sequencing and PCR methods you are using).

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 degenerated positions (multi-state characters) that anneal to sequences found in the alignment down to that level.

Max Difference in Tm (°C): The maximum difference between forward and reverse primer annealing temperature that you can accept.

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 SM, Leitner T, Barron AE. 2012. Quantitative experimental determination of primer-dimer formation risk by free-solution conjugate electrophoresis. 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 SM, Leitner T, Barron AE. 2012. Quantitative experimental determination of primer-dimer formation risk by free-solution conjugate electrophoresis. Electrophoresis 33: 483-491. PMID 22331820.

Forward/Reverse Primer Options: These options allow you to specify that the 3' and/or 5' end must be a G or C.

Adaptor Options

No adaptor: No DNA handle will be attached to the construct.

De Novo Sequence Length: If you choose "No adaptor", you need to provide a de novo sequence length. This length depends on the read length of the system you are designing primers for. Different sequencing technologies have certain read length limitations. The de novo length should be the expected read length minus one primer length. In this version of PrimerDesign, the de novo length must always be longer than the Region of Interest (after gapstripping). In a future version, currently under development, this restriction will be relaxed to allow for multiple primer design for long/contig sequence analyses.

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: User input DNA handle sequences. Opens new input windows.

Tag Options

You can choose how to optimize the tag/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.

Email options

Email return will occur automatically when a computationally demanding design is requested. You may also choose "always email results", which is advisable if your internet connection is slow or server load is high. Thus, if you experience a browser timeout, select the "always email" option.

Output

Below is an example of tool output.

You may choose a pair of primers from among the options, based on your own experimental criteria. For example, you may decide that having a longer or shorter fragment serves your experiment better. Or you may wish to avoid (or include) a particular region outside the region of interest.

The primer choices provided are not guaranteed. They may require tweaking of PCR conditions, or they may fail to amplify. They are optimized to the calculated entropy, complexity, and Tm of your DNA, but other factors may affect your results.

Links

Return to Primer Design tool interface.

 

Questions or comments? Contact us at seq-info@lanl.gov.

 
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