Microchip-based electrophoretic analysis of low abundant unknown mutations using site-specific nicking and ligation enzymes

Rondedrick D Sinville, Shawn Llopis, Hanna Pincas, Francis Barany, Steven A. Soper

Research output: Contribution to conferencePaperpeer-review

Abstract

A polymer-based microfluidic device was used to electrophoretically sort enzymatically cleaved and/or resealed fluorescently-labeled oligonucleotide products generated from an allele-specific ligase detection reaction (LDR) and/or endonulease V (EndoV)/LDR mutation scanning assay. Analyses were conducted using microchannels formed via hot embossing of LiGA-generated high aspect ratio microstructures, which were subsequently filled with linear polyacrylamide (LPA). These separations were aided by the use of a novel surface modification procedure developed for poly (methyl methacrylate) (PMMA), which minimized the electroosmotic flow (EOF) and nonspecific analyte-wall interactions; it was shown to provide increases in peak efficiencies (>1.3×106 m-1) compared to pristine surfaces (∼5×105 m-1). LDR products indicative of colorectal cancer point mutations were successfully separated in ∼4 min, while unknown p53 mutations were elucidated using EndoV with a subsequent reduction in noise upon resealing of incorrectly nicked DNA using ligation.

Original languageEnglish (US)
Pages1450-1452
Number of pages3
StatePublished - Jan 1 2006
Event10th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2006 - Tokyo, Japan
Duration: Nov 5 2006Nov 9 2006

Other

Other10th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2006
Country/TerritoryJapan
CityTokyo
Period11/5/0611/9/06

Keywords

  • Endonuclease V
  • Ligase detection reaction
  • Microchip electrophoresis

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