title: A primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP).
abstract: There are various ways that priming can occur in nucleic acid amplification reactions. While most reactions rely on a primer to initiate amplification, a mechanism for DNA amplification has been developed in which hairpin sequences at the 3' terminus of a single-stranded oligonucleotide fold on themselves to initiate priming. Unfortunately, this method is less useful for diagnostic applications because the self-folding efficiency is low and only works over a narrow range of reaction temperatures. In order to adapt this strategy for analytical applications we have developed a variant that we term phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP). In PS-THSP a phosphorothioate (PS) modification is incorporated into the DNA backbone, leading to a reduction in the thermal stability of dsDNA and increased self-folding of terminal hairpins. By optimizing the number of PS linkages that are included in the initial template, we greatly increased self-folding efficiency and the range of reaction temperatures, ultimately achieving a detection limit of 1 pM. This improved method was readily adapted to the detection of single nucleotide polymorphisms and to the detection of non-nucleic acid analytes, such as alkaline phosphatase, which was quantitatively detected at a limit of 0.05 mU/mL, approximately 10-fold better than commercial assays. Graphical abstract Efficient self-folding by phosphorothioate (PS) modification.
authors.affiliation: Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, University of Texas at Austin, 2500 Speedway MBB 3.424, Austin, TX, 78712, USA.
authors.forename: Cheulhee
authors.initials: C
authors.lastname: Jung
authors.affiliation: Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, University of Texas at Austin, 2500 Speedway MBB 3.424, Austin, TX, 78712, USA. ellingtonlab@gmail.com.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D055029:Inverted Repeat Sequences; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D057089:Pathology, Molecular / Q000379:methods*; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide
chemical_list: D017931:DNA Primers; D054735:Phosphorothioate Oligonucleotides; D004247:DNA; D000469:Alkaline Phosphatase
keywords: Isothermal amplification; PS-THSP; Phosphorothioate; Self-folding