title: A high-throughput assay for the comprehensive profiling of DNA ligase fidelity.
abstract: DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.
authors.affiliation: New England BioLabs, Inc., Ipswich, MA 01938-2723, USA lohman@neb.com.
authors.forename: Gregory J S
authors.initials: GJ
authors.lastname: Lohman
authors.affiliation: New England BioLabs, Inc., Ipswich, MA 01938-2723, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Bauer
authors.affiliation: New England BioLabs, Inc., Ipswich, MA 01938-2723, USA.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Nichols
authors.affiliation: New England BioLabs, Inc., Ipswich, MA 01938-2723, USA.
authors.forename: Laurie
authors.initials: L
authors.lastname: Mazzola
authors.affiliation: New England BioLabs, Inc., Ipswich, MA 01938-2723, USA.
authors.forename: Joanna
authors.initials: J
authors.lastname: Bybee
authors.affiliation: New England BioLabs, Inc., Ipswich, MA 01938-2723, USA.
authors.forename: Danielle
authors.initials: D
authors.lastname: Rivizzigno
authors.affiliation: New England BioLabs, Inc., Ipswich, MA 01938-2723, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Cantin
authors.affiliation: New England BioLabs, Inc., Ipswich, MA 01938-2723, USA evanst@neb.com.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Evans
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D020137:Base Pair Mismatch; D020029:Base Pairing; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000302:isolation & purification; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006863:Hydrogen-Ion Concentration; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013696:Temperature; D016963:Thermus thermophilus / Q000737:chemistry* / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D019793:Fluorescein