Document Detail


Detection of telomerase activity in high concentration of cell lysates using primer-modified gold nanoparticles.
MedLine Citation:
PMID:  20932008     Owner:  NLM     Status:  In-Process    
Abstract/OtherAbstract:
Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its use has been significantly limited when performed directly in complex, interferant-laced samples. In this work, we report a modification of the TRAP assay that allows the detection of high-fidelity amplification of telomerase products directly from concentrated cell lysates. Briefly, we covalently attached 12 nm gold nanoparticles (AuNPs) to the telomere strand (TS) primer, which is used as a substrate for telomerase elongation. These TS-modified AuNPs significantly reduce polymerase chain reaction (PCR) artifacts (such as primer dimers) and improve the yield of amplified telomerase products relative to the traditional TRAP assay when amplification is performed in concentrated cell lysates. Specifically, because the TS-modified AuNPs eliminate most of the primer-dimer artifacts normally visible at the same position as the shortest amplified telomerase PCR product apparent on agarose gels, the AuNP-modified TRAP assay exhibits excellent sensitivity. Consequently, we observed a 10-fold increase in sensitivity for cancer cells diluted 1000-fold with somatic cells. It thus appears that the use of AuNP-modified primers significantly improves the sensitivity and specificity of the traditional TRAP assay and may be an effective method by which PCR can be performed directly in concentrated cell lysates.
Authors:
Yi Xiao; Karen Y Dane; Takanori Uzawa; Andrew Csordas; Jiangrong Qian; H Tom Soh; Patrick S Daugherty; Eric T Lagally; Alan J Heeger; Kevin W Plaxco
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Journal of the American Chemical Society     Volume:  132     ISSN:  1520-5126     ISO Abbreviation:  J. Am. Chem. Soc.     Publication Date:  2010 Nov 
Date Detail:
Created Date:  2010-10-28     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  7503056     Medline TA:  J Am Chem Soc     Country:  United States    
Other Details:
Languages:  eng     Pagination:  15299-307     Citation Subset:  IM    
Affiliation:
Department of Physics, Materials Department, and Institute for Polymers and Organic Solids, University of California, Santa Barbara, California 93106, United States.
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