Document Detail


Incorporation of a viral DNA-packaging motor channel in lipid bilayers for real-time, single-molecule sensing of chemicals and double-stranded DNA.
MedLine Citation:
PMID:  23348364     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Over the past decade, nanopores have rapidly emerged as stochastic biosensors. This protocol describes the cloning, expression and purification of the channel of the bacteriophage phi29 DNA-packaging nanomotor and its subsequent incorporation into lipid membranes for single-pore sensing of double-stranded DNA (dsDNA) and chemicals. The membrane-embedded phi29 nanochannel remains functional and structurally intact under a range of conditions. When ions and macromolecules translocate through this nanochannel, reliable fingerprint changes in conductance are observed. Compared with other well-studied biological pores, the phi29 nanochannel has a larger cross-sectional area, which enables the translocation of dsDNA. Furthermore, specific amino acids can be introduced by site-directed mutagenesis within the large cavity of the channel to conjugate receptors that are able to bind specific ligands or analytes for desired applications. The lipid membrane-embedded nanochannel system has immense potential nanotechnological and biomedical applications in bioreactors, environmental sensing, drug monitoring, controlled drug delivery, early disease diagnosis and high-throughput DNA sequencing. The total time required for completing one round of this protocol is around 1 month.
Authors:
Farzin Haque; Jia Geng; Carlo Montemagno; Peixuan Guo
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, N.I.H., Extramural     Date:  2013-01-24
Journal Detail:
Title:  Nature protocols     Volume:  8     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-01-25     Completed Date:  2013-06-19     Revised Date:  2014-01-09    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  373-92     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Bacillus Phages / genetics,  metabolism*
DNA / isolation & purification*
DNA Packaging / physiology*
Lipid Bilayers / metabolism*
Models, Molecular
Molecular Motor Proteins / metabolism*
Mutagenesis, Site-Directed / methods
Nanopores*
Nanotechnology / methods*
Grant Support
ID/Acronym/Agency:
EB012135/EB/NIBIB NIH HHS; R01 EB012135/EB/NIBIB NIH HHS
Chemical
Reg. No./Substance:
0/Lipid Bilayers; 0/Molecular Motor Proteins; 9007-49-2/DNA
Comments/Corrections

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