Help:Primers/Design

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General guidelines • Constructing new BioBrick parts

General guidelines for primer design

Primer design can either be done by hand or using software tools. We've included a list of general guidelines for use when designing new primers.

If you are interested in sequencing or amplifying a BioBrick part or plasmid, please see the catalog of commonly used BioBrick primer sequences. These primers have been successfully been used by others, so where possible, we suggest that you reuse existing primers.

• Primers are always specified 5' to 3', left to right. Verify that your primers are designed and ordered in the correct orientation.
• For sequencing and PCR applications, primer should
  • be 20-30 nucleotides in length.
  • have a GC content of 40-60%
  • have a melting temp (T_m) of 55-65°C
• The 3' end of the primer should be an exact match to the template DNA, because extension by DNA polymerase, during PCR, depends on a good match at the 3' end. Thus, primers are often designed with the 3' end of the primer as a G or C. G-C base pairs have a stronger bond than A-T base pairs (3 hydrogen bonds versus 2).
• Conversely, 5' tails can be readily added to primers without impacting primer annealing. For example, to construct new BioBrick parts, you'll likely need to add BioBrick prefix and suffix sequences to your forward and reverse primers.
• Primers should not have palindromes or inverted repeat sequences. Such sequences cause primers to either dimerize or form hairpins that interfere with proper annealing to the template.
  • You can check for self-annealing and hairpins using software tools. You want to avoid structures with ΔG < -5 kcal/mol
• Primer pairs should
  • have similar annealing temperatures
  • not be complementary to one another
• Avoid runs of over 3 nucleotides (i.e., CCCC). Long stretches of G, in particular, give problems.

Primer design isn't perfect. Sometimes you can design what looks like a great primer and yet the PCR or sequencing just fails. While there are different reasons that a PCR can fail, Tom Knight's rule of thumb is that if a PCR fails, try it again but work a bit harder by varying the annealing temperature or something else. If it fails again, redesign your primers.

Primers longer than 50 nucleotides

• Chemical synthesis of primers occurs via sequential addition of bases from 3' to 5', and addition of each base is approximately 99% efficient. Thus, when you receive a synthesized oligo, only 0.99^n-1 of the molecules will be correct, where n is the primer length. For example, for a primer of length 70 nucleotides, only 50% of the molecules will be correct. Errors in primer synthesis include truncations, internal deletions, and mutated bases. Truncated primers or primers with internal deletions can be eliminated via purification by HPLC or PAGE. Such purification steps are available from most oligo synthesis companies, but do substantially increase primer cost. For primers over over ~60 nucleotides in length or for sensitive applications, such as terminator construction, you might consider paying for an extra purification step when ordering a primer.

Primers for TA cloning of PCR products

• If you are designing primers for PCR and you plan to clone the PCR product using [http://openwetware.org/wiki/Knight:TOPO_TA_cloning TA or TOPO-TA cloning], the 5' end of the PCR primer has a strong effect on the likelihood of 3' A addition to the PCR product when using Taq or Taq mixtures as enzymes during PCR Brownstein. To favor addition of a 3' A overhang on the PCR products and facilitate TA cloning, add the sequence GTTTCTT to the 5' end of the primer.

Primers for restriction digest of PCR products

• If you are designing primers for PCR and you plan to digest the PCR product with restriction enzymes afterwards, note that some enzymes do not cut efficiently near the ends of linear DNA. To be safe, include 8 extra bases on the 5' end of your primers to increase the efficiency of cutting.
  • NEB has a table of how many "spacer" bases are needed for cleavage near DNA ends NEB. They report that many restriction enzymes reportedly work with 4 bases, but in the fine print, they recommend increasing the numbers listed in the table by 4 for efficient cutting.
  • As an example, Reshma Shetty found that XhoI required more than 4 bases (8 bases was used successfully).

Primers for constructing BioBrick parts via PCR

Enter the sequence of your new part in the Registry.

If you are constructing a protein coding sequence, do the following

1. Design a forward primer to your new BioBrick part comprised of the BioBrick protein coding prefix sequence GTT TCT TCG AAT TCG CGG CCG CTT CTA G followed by the first 20-30 nucleotides of the protein coding region, beginning with the ATG
2. Design a reverse primer to your new BioBrick part comprised of the last 20-30 or so nucleotides of the protein coding sequence, excluding the stop codon, followed by the BioBrick protein coding suffix sequence 5'-TAA TAA TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'. Note that the BioBrick protein coding suffix sequence includes the double stop codon TAA TAA. (Note: In some cases, for example if you wish use your part directly for making protein fusions using BioScaffold parts [1] or make your part more versatile by giving other users the capacity to do so in the future, then you should only include one stop codon TAA or make a version of your part that has only one stop codon. In this case your sequence should be followed by the BioScaffold and BioBrick compatible protein suffix 5'-TAA TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3' instead of the BioBrick protein coding suffix sequnce. Since the scar region that is created by the composition of parts, also introduces a stop codon after protein parts, including only one stop codon within the frame of the part may be sufficient (especially if your part will be followed by a subsequent part.))
3. Be sure and follow the guidelines above for design of the primer sequence that is complementary to the template.
4. Then take the reverse complement of your reverse primer.

If you are constructing any other kind of BioBrick part besides a protein coding region, do the following

1. Design a forward primer to your new BioBrick part comprised of the BioBrick prefix sequence 5'-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG-3' followed by the first 20-30 or so nucleotides of the part sequence.
2. Design a reverse primer to your new BioBrick part comprised of the last 20-30 or so nucleotides of the part sequence followed by the BioBrick suffix sequence 5'-TAC TAG TAG CGG CCG CTG CAG GAA GAA AC-3'
3. Be sure and follow the guidelines above for design of the primer sequence that is complementary to the template.
4. Then take the reverse complement of your reverse primer.

You can find a good tutorial explaining where these prefix and suffix sequences come from [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication here].

References

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1. Brownstein pmid=8780871
2. NEB [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp NEB's page on Cleavage Close to the End of DNA Fragments (linearized vector)]

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