Affordable Access

deepdyve-link
Publisher Website

Robust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons.

Authors
  • Jiang, Yu Sherry
  • Bhadra, Sanchita
  • Li, Bingling
  • Wu, Yuefeng Rose
  • Milligan, John N
  • Ellington, Andrew D
Type
Published Article
Journal
Analytical Chemistry
Publisher
American Chemical Society
Publication Date
Mar 17, 2015
Volume
87
Issue
6
Pages
3314–3320
Identifiers
DOI: 10.1021/ac504387c
PMID: 25708458
Source
Medline
License
Unknown

Abstract

Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 10(9) copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex mixture such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equivalents of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests.

Report this publication

Statistics

Seen <100 times