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Soper, Steven A.
Soper Research Group
Research Interests
  • Micro- and nano-fabrication
  • Lab-on-a-chip
  • Polymeric Microfluidic Devices


EndoV/DNA ligase mutation scanning assay using microchip capillary electrophoresis and dual-color laser-induced fluorescence detection

DC Field Value Language Kotani, Akira ko Witek, Malgorzata A. ko Osiri, John K. ko Wang, Hong ko Sinville, Rondedrick ko Pincas, Hanna ko Barany, Francis ko Soper, Steven A. ko 2014-04-10T02:36:32Z - 2013-06-17 ko 2012-01 -
dc.identifier.citation ANALYTICAL METHODS, v.4, no.1, pp.58 - 64 ko
dc.identifier.issn 1759-9660 ko
dc.identifier.uri -
dc.identifier.uri ko
dc.description.abstract We report the ability to detect with high sensitivity sporadic mutations using a mutation scanning assay, which employs thermostable endonuclease V (EndoV) and DNA ligase. The products of the mutation scanning assay were separated using microchip capillary electrophoresis (mu CE) and detected with a dual-color laser-induced fluorescence (LIF) detector. PCR products from mutant and wild-type DNA of p53 exon 8 were generated using Cy3-labeled forward and Cy5-labeled reverse primers to allow LIF detection with mCE. EndoV recognizes and primarily cleaves heteroduplexed DNA one base 30 to a mismatch and can nick matched sites at low levels as well. DNA ligase is used to reseal nicks generated at matched sites, which creates a highly sensitive and specific assay for analyzing sporadic mutations in genomic DNA. Heteroduplexed DNA samples were treated with EndoV alone and with both EndoV and DNA ligase and separated using a 4% (w/v) linear polyacrylamide gel constituted in 1x TTE buffer, 7 M urea, and 0.05% (w/v) methyl hydroxyethyl cellulose, which was used to suppress the EOF in the microchip. Sizing of the bands appearing in the electropherogram revealed the approximate position of the mutation. In this study, mutations present in p53 exon 8 generated Cy3-labeled cleavage products of 158 nt and Cy5-labeled cleavage products of 195 nt. The DNA fragments were simultaneously monitored at their respective color using a dual-color LIF system with the 158 and 195 nt fragments detected along with heteroduplexed fragments of 350 nt. The microchip separation was completed within 7 min, almost tenfold shorter time compared to conventional capillary gel electrophoresis. ko
dc.description.statementofresponsibility close -
dc.language ENG ko
dc.publisher ROYAL SOC CHEMISTRY ko
dc.subject Capillary gel electrophoresis ko
dc.subject Cleavage products ko
dc.subject DNA fragment ko
dc.subject DNA ligases ko
dc.subject DNA samples ko
dc.subject Electropherograms ko
dc.subject Endonucleases ko
dc.subject Genomic DNA ko
dc.subject High sensitivity ko
dc.subject Highly sensitive ko
dc.subject Hydroxyethyl cellulose ko
dc.subject Laser induced fluorescence ko
dc.subject Laser induced fluorescence detection ko
dc.subject Low level ko
dc.subject Microchip capillary electrophoresis ko
dc.subject Microchip separations ko
dc.subject PCR products ko
dc.subject Polyacrylamide gels ko
dc.subject Wild types ko
dc.title EndoV/DNA ligase mutation scanning assay using microchip capillary electrophoresis and dual-color laser-induced fluorescence detection ko
dc.type ARTICLE ko
dc.identifier.scopusid 2-s2.0-84862935445 ko
dc.identifier.wosid 000298883800007 ko
dc.type.rims ART ko
dc.description.wostc 7 *
dc.description.scopustc 6 * 2014-10-18 * 2014-07-12 *
dc.identifier.doi 10.1039/c1ay05366c ko
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