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Ligase detection reaction for the analysis of point mutations using free‐solution conjugate electrophoresis in a polymer microfluidic device
Author(s) -
Sinville Rondedrick,
Coyne Jennifer,
Meagher Robert J.,
Cheng YuWei,
Barany Francis,
Barron Annelise,
Soper Steven A.
Publication year - 2008
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.200800197
Subject(s) - microfluidics , conjugate , dna ligase , chromatography , chemistry , electrophoresis , polymer , materials science , nanotechnology , dna , biochemistry , organic chemistry , mathematical analysis , mathematics
We have developed a new method for the analysis of low abundant point mutations in genomic DNA using a combination of an allele‐specific ligase detection reaction (LDR) with free‐solution conjugate electrophoresis (FSCE) to generate and analyze the genetic products. FSCE eliminates the need for a polymer sieving matrix by conjugating chemically synthesized polyamide “drag‐tags” onto the LDR primers. The additional drag of the charge‐neutral drag‐tag breaks the linear scaling of the charge‐to‐friction ratio of DNA and enables size‐based separations of DNA in free solution using electrophoresis with no sieving matrix. We successfully demonstrate the conjugation of polyamide drag‐tags onto a set of four LDR primers designed to probe the K‐ras oncogene for mutations highly associated with colorectal cancer, the simultaneous generation of fluorescently labeled LDR/drag‐tag conjugate (LDR‐dt) products in a multiplexed, single‐tube format with mutant:WT ratios as low as 1:100, respectively, and the single‐base, high‐resolution separation of all four LDR‐dt products. Separations were conducted in free solution with no polymer network using both a commercial capillary array electrophoresis (CAE) system and a PMMA microchip replicated via hot‐embossing with only a Tris‐based running buffer containing additives to suppress the EOF. Typical analysis times for LDR‐dt were 11 min using the CAE system and as low as 85 s for the PMMA microchips. With resolution comparable to traditional gel‐based CAE, FSCE along with microchip electrophoresis decreased the separation time by more than a factor of 40.