Direct visualization of cystic fibrosis transmembrane regulator mutations in the clinical laboratory setting.
BACKGROUND
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.


METHODS
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.


RESULTS
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


CONCLUSIONS
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.
Strom
Charles M
CM
Nichols Institute, Quest Diagnostics, San Juan Capistrano, CA, USA.
Clark
David D
DD
Hantash
Feras M
FM
Rea
Larry
L
Anderson
Ben
B
Maul
Diana
D
Huang
Donghui
D
Traul
Donald
D
Chen Tubman
Christina
C
Garcia
Renee
R
Hess
P Patrick
PP
Wang
Hong
H
Crossley
Beryl
B
Woodruff
Evelyn
E
Chen
Rebecca
R
Killeen
Myra
M
Sun
Weimin
W
Beer
Jonathan
J
Avens
Heather
H
Polisky
Barry
B
Jenison
Robert D
RD
D001322:Autoanalysis; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D015342:DNA Probes; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012371:Robotics
C505032:CFTR protein, human; D015342:DNA Probes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator