Analytical and Bioanalytical Chemistry

, Volume 408, Issue 30, pp 8583–8591

A primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP)

Paper in Forefront

DOI: 10.1007/s00216-016-9479-y

Cite this article as:
Jung, C. & Ellington, A.D. Anal Bioanal Chem (2016) 408: 8583. doi:10.1007/s00216-016-9479-y
Part of the following topical collections:
  1. Isothermal Nucleic Acid Amplification in Bioanalysis

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

Keywords

PS-THSPSelf-foldingIsothermal amplificationPhosphorothioate

Supplementary material

216_2016_9479_MOESM1_ESM.pdf (592 kb)
ESM 1(PDF 591 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Institute for Cellular and Molecular Biology, Department of Chemistry and BiochemistryUniversity of Texas at AustinAustinUSA