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

Cheulhee Jung, Andrew D. Ellington

Research output: Contribution to journalArticle

4 Citations (Scopus)

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. [Figure not available: see fulltext.]

Original languageEnglish
Pages (from-to)8583-8591
Number of pages9
JournalAnalytical and Bioanalytical Chemistry
Volume408
Issue number30
DOIs
Publication statusPublished - 2016 Dec 1
Externally publishedYes

Keywords

  • Isothermal amplification
  • Phosphorothioate
  • PS-THSP
  • Self-folding

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

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