Microarray and microfluidic methodology for genotyping cytokine gene polymorphisms.
Cytokine genetic polymorphisms are the subject of disease-association studies that require large-scale human genotyping. Polymerase chain reaction based custom microarrays and microfluidics systems were used to develop genotyping assays for following cytokine polymorphisms: tumor necrosis factor-alpha G-308A, interleukin-4 (IL-4) C-589T, interferon-gamma (CA)n repeats, IL-1RN 86-bp variable number of tandem repeats (VNTR), and CCR5 32-bp indel. For G-308A, 70.9% of DNA samples assayed were homozygous for wild type, 25.5% were heterozygous, and none were homozygous for variant allele. For C-589T, 35.5% of DNA samples were homozygous for wild type, 38% were heterozygous, and 22% were homozygous for variant. For IL-1RN VNTR, 71% of DNA samples were homozygous and the remainder were heterozygous. For CCR5, 96.4% of amplicons were homozygous for wild type, and 3.6% were heterozygous containing deletion. For IFN-gamma (CA)n repeats, 35.6% had 2,2 alleles, 42.2% had 2,3 alleles, and 11% had 3,3 alleles with alleles 1 through 5 corresponding to 11 through 15 repeats, respectively. There was good concordance between the results we obtained and current "gold-standard" methodologies for analyzing single nucleotide polymorphisms and size polymorphisms. Electronic DNA concentration with high stringency predisposes microarray technology to hybridization fidelity and accuracy, and microfluidics systems outperform conventional methodologies for size polymorphisms. Comprehensive genotyping can be achieved for clinical epidemiologic studies on cytokine gene polymorphisms using this approach.
Sohni
Youvraj R
YR
Mayo Clinic Cancer Center Microarray Shared Resource, Rochester, MN 55905, USA. youvraj@mayo.edu
Cerhan
James R
JR
O'Kane
Dennis
D
D016207:Cytokines / Q000235:genetics*; D044085:Microfluidics / Q000379:methods*; D018895:Microsatellite Repeats; D018598:Minisatellite Repeats; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide
D016207:Cytokines