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Enhancing multi-step DNA processing by solid-phase enzyme catalysis on polyethylene glycol coated beads.

Authors
  • Li, Shaohua
  • Zhang, Aihua
  • Zatopek, Kelly
  • Parvez, Saba
  • Gardner, Andrew Fenn
  • Correa, Ivan
  • Noren, Christopher J
  • Xu, Ming-Qun
Type
Published Article
Journal
Bioconjugate Chemistry
Publisher
American Chemical Society
Publication Date
Jun 04, 2018
Identifiers
DOI: 10.1021/acs.bioconjchem.8b00299
PMID: 29864273
Source
Medline
License
Unknown

Abstract

Covalent immobilization of enzymes on solid supports provides an alternative approach to homogeneous biocatalysis by adding benefits of simple enzyme removal, improved stability, and adaptability to automation and high-throughput applications. Nevertheless, immobilized (IM) enzymes generally suffer from reduced activities compared to their soluble counterparts. The nature and hydrophobicity of the supporting material surface can introduce enzyme conformational change, spatial confinement, and limited substrate accessibility, all of which will result in the immobilized enzyme activity loss. In this work, we demonstrate through kinetic studies that flexible polyethylene glycol (PEG) moieties modifying the surface of magnetic beads improve the activ-ity of covalently-immobilized DNA replication enzymes. PEG-modified immobilized enzymes were utilized in library construction for Illumina next-generation sequencing (NGS) increasing the read coverage across AT-rich regions.

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