Designing Degenerate Primers

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Manual Method for Designing Degenerate Primers

  • Make a quick and dirty clustalx alignment (for tree of life representatives for the gene of interest if possible)
    • If using DNA, then make a DNA alignment.
    • If using a protein-coding gene, then make an amino acid alignment first and then a DNA alignment to check for base calls to reduce degeneracy later.
  • When you have your alignment:
    • Break up your alignment into ~300bp regions to try to design primers for so you will get ample coverage of your gene.
    • Mark any amino acid/nucleotide residues that are specific for your group of interest.
    • Mark any methionine (M) or tryptophan (W) residues since they have no degeneracy and are therefore good to include in primers.
    • Mark any C, D, E, F, H, K, N, Q and Y residues because they have the second least amount of degeneracy.
    • If the gene in question is part of a complex gene family and you have incorporated other genes from that gene family into your alignment for some of the same organisms as the gene in question, then check for any residues in your primer regions of interest that are the same/different for those other close genes.
  • Once primer areas of interest are identified from the above criteria, calculate degeneracy. Look for the IUPAC nucleotide code for each position in the alignment. If all organisms of interest in your area of interest have an adenine at position 426, then code for an A there. That position has no degeneracy. However, if that position could be an A for some of the organisms of interest and a T for others, then you would have to call it a W. Because a W could code for either an A or a T it has a degeneracy of 2. Once your entire section of interest is coded and you have a potential primer, multiply all of the degenerate position values together (eg. 2x2x4x2 = 32). As a rule of thumb, do not allow the total degeneracy of your primer to exceed 256.
  • To decrease degeneracy you can use an inosine (IUPAC code I) rather than an N, but usually only one or two may be used per primer or it will not work. For amino acid sequences you can also check the codon usage table for the species/genus/group/etc. of the organisms of interest and see if those genomes preferentially code a degenerate amino acid with a certain triplet. This is when it is also useful to double check your DNA alignment vs. your amino acid alignment of the same genes to see if there is reduced degeneracy from the many possibilities of certain amino acids. This is especially true for serine (S), leucine (L) and arginine (R).
  • Test the primers individually and as pairs for primer-primer interactions (see below).



Online Algorithm Method for Designing Degenerate Primers

  • Go to GeneFisher (http://bibiserv.techfak.uni-bielefeld.de/genefisher2/submission.html) and either upload a text document of your fasta-formatted sequences of interest or paste the fasta files into the online window directly.
  • Try different options for the settings of where, how long, GC content, etc. and see if any good primers result. Also, try several different fasta alignments (fewer or greater sequences, truncated sequences to be sure that they are all the same length, only sequences that are as close to the organisms you will be priming, etc.) to get different possibilities for primer pairs.
  • Test the primers individually and as pairs for primer-primer interactions (see below).



Quality Control of Primers

  • Try to avoid:
    • AT-rich regions (or anything that has a visible pattern)
    • 3 prime end dimers (they are especially bad since they completely knock out the binding of the primer to the DNA)
    • hairpins, especially ones with lots of GCs (hard to break apart)
    • any other secondary structures (primer dimers, etc.)
  • Download the free demo version of Oligo (http://www.oligo.net/oligo_post.htm).
    • Open the Oligo 6 demo icon and then follow these instructions: File tab, then open, select test database and hit ok, then under the new test database window menu select edit and then select add. Type in the name and 5-3 prime sequence of your proposed primer. Check the 3 prime dimer energy (0-3 is best, ~3~6 is acceptable and anything greater than that will not work because additional heat will probably destroy other parts of your gene before it breaks up the primer dimer with itself. This only gives you a primer self check. You cannot import your own gene or your primers into the sequence analysis in order to check for primers working together and any problems they might have.
  • To check for primer-primer interactions go to OligoCalc. Enter your primers together in a string from 5-3 prime with ~20 single nucleotides (eg. AAAAAA, etc.; multiple Ns will not work) between them. Hit the Calculate button and check for any bad interactions between the two sides primers. This allows you to trick the program by seemingly entering in one primer, when it is actually two but with enough dead space inbetween them that folding and interactions will be picked up if they were to occur.



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