Rapid KRAS Mutation Detection via Hybridization-Induced Aggregation of Microbeads.
Using hybridization-induced aggregation (HIA), a unique bead-based DNA detection technology scalable for a microchip platform, we describe a simplistic, low-cost method for rapid mutation testing. HIA utilizes a pair of sequence-specific oligonucleotide probes bound to magnetic microbeads. Hybridization to a target DNA strand tethers the beads together, inducing bead aggregation. By simply using the extent of bead aggregation as a measure of the hybridization efficiency, we avoid the need for additional labels and sophisticated analytical equipment. Through strategic manipulation of the assay design and experimental parameters, we use HIA to facilitate, for the first time, the detection of single base mutations in a gene segment and, specifically, the detection of activating KRAS mutations. Following the development and optimization of the assay, we apply it for KRAS mutation analysis of four human cancer cell lines. Ultimately, we present a proof-of-principle method for detecting any of the common KRAS mutations in a single-step, 2 min assay, using only one set of oligonucleotide probes, for a total analysis time of less than 10 min post-PCR. The assay is performed at room temperature and uses simple, inexpensive instrumentation that permits multiplexed analysis.
Department of Chemistry, University of Virginia , Charlottesville, Virginia 22904, United States.
Hillary S
HS
Sloane
Department of Biomedical Engineering, University of Virginia , Charlottesville, Virginia 22908, United States.|Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine , Charlottesville, Virginia 22908, United States.
Kimberly A
KA
Kelly
Department of Chemistry, University of Virginia , Charlottesville, Virginia 22904, United States.|Department of Pathology, University of Virginia , Charlottesville, Virginia 22908, United States.|Department of Mechanical Engineering, University of Virginia , Charlottesville, Virginia 22904, United States.
James P
JP
Landers
D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D060328:Magnetic Phenomena; D008863:Microspheres; D009154:Mutation / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D013997:Time Factors
C117307:KRAS protein, human; D015345:Oligonucleotide Probes; D016283:Proto-Oncogene Proteins p21(ras)