Microsphere bead arrays and sequence validation of 5/7/9T genotypes for multiplex screening of cystic fibrosis polymorphisms.
The development of simple and rapid methods for the detection of the common genetic mutations associated with cystic fibrosis (CF) requires access to positive-control samples including the 5/7/9T variants of intron 8. We used PCR and a simple multiplex bead-array assay to identify 5/7/9T control samples from 29 commercially available DNA samples. Unpurified PCR products were directly hybridized to color-coded beads containing allele-specific capture probes for 5/7/9T detection. The performance of the assay was investigated using reverse-complement oligonucleotides, individual PCR products, and multiplex PCR products for 5/7/9T detection within a complex CFTR screening assay. Samples were genotyped by grouping the relative signal intensities from each capture probe. Of 29 commercially available DNA samples analyzed, 2 5T/7T, 2 5T/9T, 9 7T/9T, 11 7T/7T, and 5 9T/9T genotypes were identified. The genotype within each sample group was confirmed by DNA sequencing. The assay was compatible with the analysis of 10 to 1000 ng of genomic DNA isolated from whole blood and allowed for the separate identification of primary CFTR mutations from reflex variants. The correct identification of positive controls demonstrated the utility of a simple bead-array assay and provided accessible samples for assay optimization and for routine quality control in the clinical laboratory.
Hadd
Andrew G
AG
Ambion Diagnostics, 2170 Woodward Street, Austin, TX 78744, USA. ahadd@ambion.com.
Laosinchai-Wolf
Walairat
W
Novak
Chris R
CR
Badgett
Marty R
MR
Isgur
Lesley A
LA
Goldrick
Marianna
M
Walkerpeach
Cindy R
CR
D000483:Alleles; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D005787:Gene Frequency; D005821:Genetic Techniques; D005820:Genetic Testing; D005838:Genotype; D006801:Humans; D007438:Introns; D008863:Microspheres; D009154:Mutation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA
C505032:CFTR protein, human; D009841:Oligonucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA