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Simultaneous detection of 19 K ‐ras mutations by free‐solution conjugate electrophoresis of ligase detection reaction products on glass microchips
Author(s) -
Albrecht Jennifer Coyne,
Kotani Akira,
Lin Jennifer S.,
Soper Steven A.,
Barron Annelise E.
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200462
Subject(s) - electrophoresis , chromatography , chemistry , analytical chemistry (journal) , detection limit , conjugate , resolution (logic) , microfluidics , electric field , gel electrophoresis , materials science , nanotechnology , physics , biochemistry , computer science , mathematics , mathematical analysis , artificial intelligence , quantum mechanics
We demonstrate here the power and flexibility of free‐solution conjugate electrophoresis ( FSCE ) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction ( LDR ) to detect point mutations, even at low abundance compared to the wild‐type DNA . Here, four large drag‐tags are used to achieve free‐solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K ‐ras oncogene. LDR ‐ FSCE enabled electrophoretic resolution of these 19 LDR ‐ FSCE products by CE in 13.5 min ( E = 310 V/cm) and by microchip electrophoresis in 140 s ( E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free‐solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply ( E = 700 V/cm), and the 19 LDR ‐ FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K ‐ ras mutations on integrated “sample‐in/answer‐out” devices with amplification, LDR , and detection all on one platform.