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 language | English |
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Pages (from-to) | 8583-8591 |
Number of pages | 9 |
Journal | Analytical and Bioanalytical Chemistry |
Volume | 408 |
Issue number | 30 |
DOIs | |
Publication status | Published - 2016 Dec 1 |
Externally published | Yes |
Bibliographical note
Publisher Copyright:© 2016, Springer-Verlag Berlin Heidelberg.
Keywords
- Isothermal amplification
- PS-THSP
- Phosphorothioate
- Self-folding
ASJC Scopus subject areas
- Analytical Chemistry
- Biochemistry