Ligase detection reaction for the analysis of point mutations using free-solution conjugate electrophoresis in a polymer microfluidic device.
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.
Sinville
Rondedrick
R
Center for BioModular Multi-Scale Microsystems, Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA.
Coyne
Jennifer
J
Meagher
Robert J
RJ
Cheng
Yu-Wei
YW
Barany
Francis
F
Barron
Annelise
A
Soper
Steven A
SA
D001483:Base Sequence; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods; D046209:Electrophoresis, Microchip / Q000379:methods*; D011905:Genes, ras; D005838:Genotype; D006801:Humans; D022521:Ligase Chain Reaction / Q000379:methods*; D017354:Point Mutation; D019904:Polymethyl Methacrylate
D017931:DNA Primers; D004273:DNA, Neoplasm; D019904:Polymethyl Methacrylate