Direct Visualization of Cystic Fibrosis Transmembrane Regulator Mutations in the Clinical Laboratory Setting

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2004-05, Vol.50 (5), p.836-845
• Main Authors: Strom, Charles M, Clark, David D, Hantash, Feras M, Rea, Larry, Anderson, Ben, Maul, Diana, Huang, Donghui, Traul, Donald, Chen Tubman, Christina, Garcia, Renee, Hess, P. Patrick, Wang, Hong, Crossley, Beryl, Woodruff, Evelyn, Chen, Rebecca, Killeen, Myra, Sun, Weimin, Beer, Jonathan, Avens, Heather, Polisky, Barry, Jenison, Robert D
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Autoanalysis; Automation; Biological and medical sciences; Biotin; Cystic fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Deoxyribonucleic acid; DNA; DNA Probes; Fundamental and applied biological sciences. Psychology; Genetic testing; Genetic Testing - methods; Genetics; Genotype; Genotype & phenotype; Humans; Hybridization; Information systems; Investigative techniques, diagnostic techniques (general aspects); Laboratory tests; Medical laboratories; Medical sciences; Mutation; Oligonucleotide Array Sequence Analysis - methods; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Polymorphism; Polymorphism, Single Nucleotide; Probes; Robotics; Silicon wafers; Software; Thin films
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2003.026088

The recommendation for population- based cystic fibrosis (CF) carrier screening by the American College of Medical Genetics for the 25 most prevalent mutations and 6 polymorphisms in the CF transmembrane regulatory gene has greatly increased clinical laboratory test volumes. We describe the development and technical validation of a DNA chip in a 96-well format to allow for high-throughput genotype analysis. The CF Portrait chip contains an 8 x 8 array of capture probes and controls to detect all requisite alleles. Single-tube multiplex PCR with 15 biotin-labeled primer pairs was used to amplify sequences containing all single-nucleotide polymorphisms to be interrogated. Detection of a thin-film signal created by hybridization of multiplex PCR-amplified DNA to complementary capture probes was performed with an automated image analysis instrument, NucleoSight. Allele classification, data formatting, and uploading to a laboratory information system were fully automated. The described platform correctly classified all mutations and polymorphisms and can screen approximately 1300 patient samples in a 10-h shift. Final validation was performed by two separate 1000-sample comparisons with Roche CF Gold line probe strips and the Applera CF OLA, Ver 3.0. The CF Portrait Biochip made no errors during this validation, whereas the Applera assay made seven miscalls of the IVS-8 5T/7T/9T polymorphism The CF Portrait platform is an automated, high-throughput, DNA chip-based assay capable of accurately classifying all CF mutations in the recommended screening panel, including the IVS-8 5T/7T/9T polymorphism.