A primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP)
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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.
KeywordsPS-THSP Self-folding Isothermal amplification Phosphorothioate
This work was supported by the National Institutes of Health [5 R01 AI092839, 5 R01 GM094933]. We sincerely thank Caitlin Sanford for her editing services.
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Conflict of interest
The authors declare that they have no competing interests.
- 10.Jung C, Chung JW, Kim UO, Kim MH, Park HG. Isothermal target and signaling probe amplification method, based on a combination of an isothermal chain amplification technique and a fluorescence resonance energy transfer cycling probe technology. Anal Chem. 2010;82:5937–43. doi: 10.1021/ac100606m.CrossRefGoogle Scholar
- 19.LaPlanche LA, James TL, Powell C, et al. Phosphorothioate-modified oligodeoxyribonucleotides. III. NMR and UV spectroscopic studies of the Rp-Rp, Sp-Sp, and Rp-Sp duplexes, [d(GGSAATTCC)]2, derived from diastereomeric O-ethyl phosphorothioates. Nucleic Acids Res. 1986;14:9081–93.CrossRefGoogle Scholar