title: Chemoselective Coupling Preserves the Substrate Integrity of Surface-Immobilized Oligonucleotides for Emulsion PCR-Based Gene Library Construction.
abstract: Combinatorial bead libraries figure prominently in next-generation sequencing and are also important tools for in vitro evolution. The most common methodology for generating such bead libraries, emulsion PCR (emPCR), enzymatically extends bead-immobilized oligonucleotide PCR primers in emulsion droplets containing a single progenitor library member. Primers are almost always immobilized on beads via noncovalent biotin-streptavidin binding. Here, we describe covalent bead functionalization with primers (∼106primers/2.8-μm-diameter bead) via either azide-alkyne click chemistry or Michael addition. The primers are viable polymerase substrates (4-7% bead-immobilized enzymatic extension product yield from one thermal cycle). Carbodiimide-activated carboxylic acid beads only react with oligonucleotides under conditions that promote nonspecific interactions (low salt, low pH, no detergent), comparably immobilizing primers on beads, but yielding no detectable enzymatic extension product. Click-functionalized beads perform satisfactorily in emPCR of a site-saturation mutagenesis library, generating monoclonal templated beads (104-105copies/bead, 1.4-kb amplicons). This simpler, chemical approach to primer immobilization may spur more economical library preparation for high-throughput sequencing and enable more complex surface elaboration for in vitro evolution.
authors.affiliation: Department of Chemistry, The Scripps Research Institute , 130 Scripps Way, Jupiter, Florida 33458, United States.
authors.forename: Marie L
authors.identifier: 0000-0003-2437-7550
authors.initials: ML
authors.lastname: Malone
authors.affiliation: Department of Chemistry, The Scripps Research Institute , 130 Scripps Way, Jupiter, Florida 33458, United States.
authors.forename: Valerie J
authors.initials: VJ
authors.lastname: Cavett
authors.affiliation: Department of Chemistry, The Scripps Research Institute , 130 Scripps Way, Jupiter, Florida 33458, United States.
authors.forename: Brian M
authors.identifier: 0000-0002-6531-6693
authors.initials: BM
authors.lastname: Paegel
mesh_terms: D002234:Carbodiimides / Q000737:chemistry; D002264:Carboxylic Acids / Q000737:chemistry; D057930:Click Chemistry; D020650:Combinatorial Chemistry Techniques; D017931:DNA Primers; D004655:Emulsions / Q000737:chemistry; D015723:Gene Library; D007202:Indicators and Reagents; D007700:Kinetics; D016296:Mutagenesis; D009841:Oligonucleotides / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D002234:Carbodiimides; D002264:Carboxylic Acids; D017931:DNA Primers; D004655:Emulsions; D007202:Indicators and Reagents; D009841:Oligonucleotides
keywords: emulsion PCR; solid-phase PCR; surface functionalization