Document Detail


DNA sequencing by denaturation: principle and thermodynamic simulations.
MedLine Citation:
PMID:  18930015     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We describe a new DNA sequencing method called sequencing by denaturation (SBD). A Sanger dideoxy sequencing reaction is performed on the templates on a solid surface to generate a ladder of DNA fragments randomly terminated by fluorescently labeled dideoxyribonucleotides. The labeled DNA fragments are sequentially denatured from the templates and the process is monitored by measuring the change in fluorescence intensities from the surface. By analyzing the denaturation profiles, the base sequence of the template can be determined. Using thermodynamic principles, we simulated the denaturation profiles of a series of oligonucleotides ranging from 12 to 32 bases and developed a base-calling algorithm to decode the sequences. These simulations demonstrate that DNA molecules up to 20 bases can be sequenced by SBD. Experimental measurements of the melting profiles of DNA fragments in solution confirm that DNA sequences can be determined by SBD. The potential limitations and advantages of SBD are discussed. With SBD, millions of sequencing reactions can be performed on a small area on a surface in parallel with a very small amount of sequencing reagents. Therefore, DNA sequencing by SBD could potentially result in a significant increase in speed and reduction in cost in large-scale genome resequencing.
Authors:
Ying-Ja Chen; Xiaohua Huang
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2008-10-07
Journal Detail:
Title:  Analytical biochemistry     Volume:  384     ISSN:  1096-0309     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2009 Jan 
Date Detail:
Created Date:  2008-11-25     Completed Date:  2009-01-23     Revised Date:  2010-09-21    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  170-9     Citation Subset:  IM    
Affiliation:
Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0412, USA.
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MeSH Terms
Descriptor/Qualifier:
DNA / chemistry*
Models, Biological
Nucleic Acid Denaturation*
Sequence Analysis, DNA / methods*
Temperature
Thermodynamics*
Grant Support
ID/Acronym/Agency:
HG003587/HG/NHGRI NIH HHS; R21 HG003587-01A1/HG/NHGRI NIH HHS; R21 HG003587-02/HG/NHGRI NIH HHS; R21 HG003587-03/HG/NHGRI NIH HHS
Chemical
Reg. No./Substance:
9007-49-2/DNA
Comments/Corrections

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