title: Kinetic characterization of on-chip DNA ligation on dendron-coated surfaces with nanoscaled lateral spacings.
abstract: We analyzed the enzymatic profiles of on-chip DNA ligation as we controlled the lateral spacing of surface-immobilized DNA substrates using dendron molecules with different sizes at the nanoscale. Enzymatic on-chip DNA ligation was performed on the dendron-coated surface within 20 min with no need for post-ligation gel electrophoresis. The enzymatic DNA repair was assessed by the fluorescence intensity at the repaired DNA duplex after thermally dissociating the unligated Cy3-labeled DNA from the DNA duplex, in which the Cy3-labeled DNA was hybridized prior to the on-chip DNA ligation. The rate of the nick-sealing reaction on the 27-acid dendron surface was 3-fold higher than that on the 9-acid dendron surface, suggesting that the wider lateral spacing determined by the larger dendron molecule could facilitate the access of DNA ligase to the nick site. The performance of on-chip DNA ligation was dropped to 10% and 3% when the nick was replaced by one- and two-nucleotide-long gaps, respectively. The 5' terminal phosphorylation of DNA strands by polynucleotide kinase and the on-chip DNA cleavage by endonucleases were also quantitatively monitored throughout the on-chip DNA ligation on the dendron-coated surface. A better understanding of the enzymatic kinetics of on-chip DNA ligation will contribute to a more reliable performance of various on-chip DNA ligation-based assays.
authors.affiliation: School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, San 31 Hyoja-dong, Pohang, 790-784, Korea.
authors.forename: Eung-Sam
authors.initials: ES
authors.lastname: Kim
authors.forename: Namgyu
authors.initials: N
authors.lastname: Lee
authors.forename: Joon Won
authors.initials: JW
authors.lastname: Park
authors.forename: Kwan Yong
authors.initials: KY
authors.lastname: Choi
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism*; D050091:Dendrimers / Q000737:chemistry*; D005821:Genetic Techniques / Q000295:instrumentation*; D007700:Kinetics; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods
chemical_list: D002232:Carbocyanines; D050091:Dendrimers; C089187:cyanine dye 3; D004247:DNA; D011088:DNA Ligases