DNA sequencing using polymerase substrate-binding kinetics

Abstract

Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications.

Single-molecule FRET reveals proofreading complexes in the large fragment of <em>Bacillus stearothermophilus</em> DNA polymerase I

“…There has been great interest in the elucidation of the mechanism by which a DNA polymerase selects the correct dNTP substrate as well as how the enzyme processes mismatched bases incorporated into the primer strand [ 17 – 20 ]. These polymerases have been employed in a variety of sequencing strategies that either involve the enzyme directly or exploit some aspect of the enzyme’s mechanism [ 21 , 22 ]. Here we have used smFRET to monitor the conformational changes involved in dNTP selection as well as conformational changes involved in the formation of a proofreading complex in the presence of mismatched bases in a primer-template substrate with Bst pol I LF.…”

Study of the genetic diversity of the aflatoxin biosynthesis cluster inAspergillussectionFlaviusing insertion/deletion markers in peanut seeds from Georgia, USA

“…Although the cost of sequencing entire genomes of microorganisms has significantly decreased in recent years (Baym et al 2015;Previte et al 2015;Rowan et al 2015), it is still impractical to sequence hundreds or thousands of individuals to generate genetic data. Here, we propose a workflow that allows prescreening the predominant aflatoxigenic strains as a means to obtain DNA sequencing information without sequencing all the individuals.…”

Wide-field TCSPC: methods and applications

“…With the increasing number of applications of photon counting techniques in different fields of science and technology, from DNA sequencing [31] to interplanetary communications [32], there is a growing need for this technology [24][25][26]33]. MCPs are now a mature technology which still continues to be used widely, but in recent years other wide-field photon counting methods have become available that show great promise, including single photon avalanche diode (SPAD) arrays and detectors based on superconducting detector technology.…”