title: Detection of CFTR mutations using ARMS and low-density microarrays.
abstract: The amplification refractory mutation system (ARMS) is routinely used for the identification of specific mutations within genomes. This PCR-based assay, although simple, is performed at a low-throughput scale, usually requiring gel-electrophoresis for the identification of specific mutations. We have applied the ARMS technology to a low-density microarray system to facilitate the needs of the medical clinic; high-throughput capabilities and ease-of-use. Mutations within the cystic fibrosis transmembrane regulator (CFTR) gene (DeltaF508, 1717-1G>A, G542X, 621+1G>T, and N1303K) were detected by multiplex-ARMS-PCR, and fragments were post-PCR labeled with Cy5. Amine-modified probes specific for both the wild-type and mutant forms of each mutation site were attached to glass substrates. Following hybridization of the PCR fragments to the attached probes (in a low-density microarray format), confirmation of the presence of specific sequences was achieved using a commercial scanner, as well as a fabricated low-cost fluorescent detector and applicable software. The novel combination of the ARMS and low-density microarray technologies allows for a high-throughput, simple means to rapidly identify multiple known mutations for many genetic diseases including cystic fibrosis.
authors.affiliation: Healthspex Corp., 9111 Cross Park Drive, E-119, Knoxville, TN 37923, USA. shannon@healthspex.com
authors.forename: Shannon
authors.initials: S
authors.lastname: Eaker
authors.forename: Matt
authors.initials: M
authors.lastname: Johnson
authors.forename: Josh
authors.initials: J
authors.lastname: Jenkins
authors.forename: Martin
authors.initials: M
authors.lastname: Bauer
authors.forename: Stephen
authors.initials: S
authors.lastname: Little
mesh_terms: D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000032:analysis* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014584:User-Computer Interface
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator