DNA sequencing using polymerase substrate-binding kinetics.

Michael John Robert Previte,Christian Gloeckner,John Vieceli,Matthew Kellinger,Cheng-Yao Chen,Ali Nikoomanzar,Rigo Pantoja,Mostafa Ronaghi,Amirali Kia,Sergey Etchin,Beibei Wang,Erin Bomati,Jin Shi,Molly Min He,Chunhong Zhou

doi:10.1038/ncomms6936

Michael John Robert Previte, Christian Gloeckner + Show 13 more

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https://doi.org/10.1038/ncomms6936

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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.

Highlights

Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing
For the concept of sequencing using polymerase/DNA-binding kinetics (SPDBK), we propose the following simplified kinetic model
The first step in the kinetic pathway described in equation (1) is the reversible binding of the polymerase to the DNA, k1, which is concerted or followed by the nucleotide substrate binding to the enzyme/DNA complex to form a tertiary complex, k2

Summary

Introduction

Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. 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. The combination of co-crystal structures of DNA polymerase in action[13] and fast mixing approaches, such as rapid-quench flow and stoppedflow[14,15], has provided unprecedented kinetics and mechanistic insights into polymerase function[16] These studies have shown that in the presence of a correct nucleotide, DNA polymerase forms a stable ‘closed’ ternary complex with a DNA template and the nucleotide, leading to nucleotide incorporation[17]. It can be inferred from these observations that the binding kinetics of polymerase to DNA template in the presence of correct and mismatch nucleotides can be discriminated and subsequently used for DNA sequencing

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