title: Quantitative experimental determination of primer-dimer formation risk by free-solution conjugate electrophoresis.
abstract: DNA barcodes are short, unique ssDNA primers that "mark" individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 base-pairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive base-pairs formed, yet non-consecutive base-pairs did not create stable dimers even when 20 out of 30 possible base-pairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation.
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA 94305-5444, USA. sdesmar@stanford.edu
authors.forename: Samantha M
authors.initials: SM
authors.lastname: Desmarais
authors.forename: Thomas
authors.initials: T
authors.lastname: Leitner
authors.forename: Annelise E
authors.initials: AE
authors.lastname: Barron
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000592:standards; D024202:Electrophoretic Mobility Shift Assay; D005456:Fluorescent Dyes / Q000737:chemistry; D008969:Molecular Sequence Data; D034444:Peptoids / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D034444:Peptoids