title: Exquisite allele discrimination by toehold hairpin primers.
abstract: The ability to detect and monitor single nucleotide polymorphisms (SNPs) in biological samples is an enabling research and clinical tool. We have developed a surprising, inexpensive primer design method that provides exquisite discrimination between SNPs. The field of DNA computation is largely reliant on using so-called toeholds to initiate strand displacement reactions, leading to the execution of kinetically trapped circuits. We have now similarly found that the short toehold sequence to a target of interest can initiate both strand displacement within the hairpin and extension of the primer by a polymerase, both of which will further stabilize the primer:template complex. However, if the short toehold does not bind, neither of these events can readily occur and thus amplification should not occur. Toehold hairpin primers were used to detect drug resistance alleles in two genes, rpoB and katG, in the Mycobacterium tuberculosis genome, and ten alleles in the Escherichia coli genome. During real-time PCR, the primers discriminate between mismatched templates with Cq delays that are frequently so large that the presence or absence of mismatches is essentially a 'yes/no' answer.
authors.affiliation: Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Byrom
authors.affiliation: Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Sanchita
authors.initials: S
authors.lastname: Bhadra
authors.affiliation: Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Yu Sherry
authors.initials: YS
authors.lastname: Jiang
authors.affiliation: Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA ellingtonlab@gmail.com.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
mesh_terms: D000483:Alleles; D017931:DNA Primers / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D009169:Mycobacterium tuberculosis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
chemical_list: D017931:DNA Primers