Document Detail

A simple method for amplifying RNA targets (SMART).
MedLine Citation:
PMID:  22691910     Owner:  NLM     Status:  MEDLINE    
We present a novel and simple method for amplifying RNA targets (named by its acronym, SMART), and for detection, using engineered amplification probes that overcome existing limitations of current RNA-based technologies. This system amplifies and detects optimal engineered ssDNA probes that hybridize to target RNA. The amplifiable probe-target RNA complex is captured on magnetic beads using a sequence-specific capture probe and is separated from unbound probe using a novel microfluidic technique. Hybridization sequences are not constrained as they are in conventional target-amplification reactions such as nucleic acid sequence amplification (NASBA). Our engineered ssDNA probe was amplified both off-chip and in a microchip reservoir at the end of the separation microchannel using isothermal NASBA. Optimal solution conditions for ssDNA amplification were investigated. Although KCl and MgCl(2) are typically found in NASBA reactions, replacing 70 mmol/L of the 82 mmol/L total chloride ions with acetate resulted in optimal reaction conditions, particularly for low but clinically relevant probe concentrations (≤100 fmol/L). With the optimal probe design and solution conditions, we also successfully removed the initial heating step of NASBA, thus achieving a true isothermal reaction. The SMART assay using a synthetic model influenza DNA target sequence served as a fundamental demonstration of the efficacy of the capture and microfluidic separation system, thus bridging our system to a clinically relevant detection problem.
Stephanie E McCalla; Carmichael Ong; Aartik Sarma; Steven M Opal; Andrew W Artenstein; Anubhav Tripathi
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2012-06-11
Journal Detail:
Title:  The Journal of molecular diagnostics : JMD     Volume:  14     ISSN:  1943-7811     ISO Abbreviation:  J Mol Diagn     Publication Date:  2012 Jul 
Date Detail:
Created Date:  2012-06-22     Completed Date:  2012-10-25     Revised Date:  2013-07-12    
Medline Journal Info:
Nlm Unique ID:  100893612     Medline TA:  J Mol Diagn     Country:  United States    
Other Details:
Languages:  eng     Pagination:  328-35     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
Center for Biomedical Engineering, School of Engineering and Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.
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MeSH Terms
Nucleic Acid Amplification Techniques / methods*
RNA / genetics*
Grant Support
1R21 A1073808-01 A1//PHS HHS
Reg. No./Substance:

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