Primer Design

Trouble-shooting Primer Applications


Contact the Corelab director for help with primer design, programmatic design, blast searching and suitability for multiplexing. MIT's Primer3 is available on-line, or via EMBOSS using eprimer3

More information on batch design and data processing methods is on our bioinformatics page.


Oligo Design Problems and Possible Solutions

Performance Problem Application Design Remedy Notes

Background due to false priming in genomic or other DNA samples with much unknown sequence.


Select oligonucleotides with high specificiy.

Determine stability (delta G) of the 3-prime ends of oligonucleotides; select only those with low or moderate stability.

Multiple PCR product bands due to false priming within and near the intended amplificationregion.


Select only oligos that will not fales prime within and near the PCR product on the target DNA.

Determine the propensity of false priming using tstability (delta G) calculations; select oligos with no strong priming affinity to any other region on the target sequence.

Background due to false priming and much of the sample is known, including related available sequence files.


Select oligos which will not false prime on known sequence other than the target sequence

False Priming Check Against Other Sequence Files: Check for false priming sites in all the sequence files selected by the user.

High background due to false priming in repetitive sequence region in the target, e.g., ALU sequences.


Select oligos which will not false prime in repetitive sequence ;-)

Check for false priming sites in known repetitive sequences downloaded and selected by the user.

Low efficiency priming because of 3-prime dimerizing and hairpin loop formation of the primer itself.


Select oligos with low dimer and hairpin formation potential at their 3-prime ends.

Check the 3-prime ends of all oligos using stability algorithms, eliminae those with dimer or hairpin potential.

Table adapted from the Oligo Primer Analysis Software User Manual (v.6), Section 12.3, p. 233

Tm and other Calculations

OSYN Primer Formulae

Oligo Calc is a very nice oligonucleotide property calculator (e.g. Tm, molecular weight, GC%, etc.)