SEQanswers

Go Back   SEQanswers > Literature Watch



Similar Threads
Thread Thread Starter Forum Replies Last Post
PubMed: De novo DNA synthesis using single-molecule PCR. Newsbot! Literature Watch 0 02-14-2012 05:00 AM
PubMed: Integrated Online System for a Pyrosequencing-Based Microbial Source Tracking Newsbot! Literature Watch 0 07-26-2011 07:30 AM
PubMed: Structural basis for the synthesis of nucleobase modified DNA by Thermus aqua Newsbot! Literature Watch 0 12-03-2010 03:00 AM
PubMed: Statistical distributions of sequencing by synthesis with probabilistic nucle Newsbot! Literature Watch 0 06-16-2009 06:00 AM
PubMed: An integrated software system for analyzing ChIP-chip and ChIP-seq data. Newsbot! Literature Watch 0 11-04-2008 06:03 AM

Reply
 
Thread Tools
Old 02-04-2010, 03:11 AM   #1
Newsbot!
RSS Posting Maniac
 

Join Date: Feb 2008
Posts: 1,443
Default PubMed: An Integrated System for DNA Sequencing by Synthesis Using Novel Nucleotide A

Syndicated from PubMed RSS Feeds

An Integrated System for DNA Sequencing by Synthesis Using Novel Nucleotide Analogues.

Acc Chem Res. 2010 Feb 3;

Authors: Guo J, Yu L, Turro NJ, Ju J

The Human Genome Project has concluded, but its successful completion has increased, rather than decreased, the need for high-throughput DNA sequencing technologies. The possibility of clinically screening a full genome for an individual's mutations offers tremendous benefits, both for pursuing personalized medicine and for uncovering the genomic contributions to diseases. The Sanger sequencing method, although enormously productive for more than 30 years, requires an electrophoretic separation step that, unfortunately, remains a key technical obstacle for achieving economically acceptable full-genome results. Alternative sequencing approaches thus focus on innovations that can reduce costs. The DNA sequencing by synthesis (SBS) approach has shown great promise as a new sequencing platform, with particular progress reported recently. The general fluorescent SBS approach involves (i) incorporation of nucleotide analogs bearing fluorescent reporters, (ii) identification of the incorporated nucleotide by its fluorescent emissions, and (iii) cleavage of the fluorophore, along with the reinitiation of the polymerase reaction for continuing sequence determination. In this Account, we review the construction of a DNA-immobilized chip and the development of novel nucleotide reporters for the SBS sequencing platform. Click chemistry, with its high selectivity and coupling efficiency, was explored for surface immobilization of DNA. The first generation (G-1) modified nucleotides for SBS feature a small chemical moiety capping the 3'-OH and a fluorophore tethered to the base through a chemically cleavable linker; the design ensures that the nucleotide reporters are good substrates for the polymerase. The 3'-capping moiety and the fluorophore on the DNA extension products, generated by the incorporation of the G-1 modified nucleotides, are cleaved simultaneously to reinitiate the polymerase reaction. The sequence of a DNA template immobilized on a surface via click chemistry is unambiguously identified with this chip-SBS system. The second generation (G-2) SBS system was developed based on the concept that the closer the structures of the added nucleotide and the primer are to their natural counterparts, the more faithfully the polymerase would incorporate the nucleotide. In this approach, the polymerase reaction is performed with the combination of 3'-capped nucleotide reversible terminators (NRTs) and cleavable fluorescent dideoxynucleotides (ddNTPs). By sacrifice of a small amount of the primers permanently terminated by ddNTPs, the majority of the primers extended by the reversible terminators are reverted to the natural ones after each sequencing cycle. We have also developed the 3'-capped nucleotide reversible terminators to solve the problem of deciphering the homopolymeric regions of the template in conventional pyrosequencing. The 3'-capping moiety on the DNA extension product temporarily terminates the polymerase reaction, which allows only one nucleotide to be incorporated during each sequencing cycle. Thus, the number of nucleotides in the homopolymeric regions are unambiguously determined using the 3'-capped NRTs. It has been established that millions of DNA templates can be immobilized on a chip surface through a variety of approaches. Therefore, the integration of these high-density DNA chips with the molecular-level SBS approaches described in this Account is expected to generate a high-throughput and accurate DNA sequencing system with wide applications in biological research and health care.

PMID: 20121268 [PubMed - as supplied by publisher]



More...
Newsbot! is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 03:49 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO