ArticleElectrophoresis2012
Quantitative experimental determination of primer-dimer formation risk by free-solution conjugate electrophoresis.
Article in Electrophoresis, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed.
- DeGenPrime provides robust primer design and optimization unlocking the biosphere.Bioinformatics advances · 2024Article
- Capillary electrophoresis applied to DNA: determining and harnessing sequence and structure to advance bioanalyses (2009-2014).Analytical and bioanalytical chemistry · 2015Review
- PrimerDesign-M: a multiple-alignment based multiple-primer design tool for walking across variable genomes.Bioinformatics (Oxford, England) · 2015Article
- A multiple-alignment based primer design algorithm for genetically highly variable DNA targets.BMC bioinformatics · 2013Article
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Authors and funding
3 authors.
Funding
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.
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Registered trials
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