Microfluidic chip-based method for genotyping microsatellites, VNTRs and insertion/deletion polymorphisms.
We have developed a method to genotype variable number of tandem repeats (VNTRs) and insertion/deletion polymorphisms using an integrated microfluidic chip-based system. We used this method to analyze a) a highly polymorphic pentanucleotide repeat (CCTTT)(n) locus within the 5'-putative promoter region of the human inducible nitric oxide synthase gene (iNOS5) which is associated with diabetic complications and infectious diseases; b) a bi-allelic 27 bp VNTR region within intron 4 of endothelial nitric oxide gene (eNOS27) which is associated with hypertension in type 2 diabetes patients with coronary heart disease and excess risk of advanced diabetic nephropathy in type 1 diabetes patients and c) an insertion/deletion polymorphism within the gene encoding angiotensin-converting enzyme (ACE/ID) which is associated with cardiovascular pathology and nitric oxide activity, and is in strong linkage disequilibrium with functional variants. Following amplifications, samples were mixed with gel-dye and markers and loaded into commercially available microfluidic chips designed for DNA sizing applications. In the study (N = 230), 95 (41%) of the DNA samples were homozygous and 135 (59%) were heterozygous for the iNOS5 repeats. For eNOS27, 173 (75%) of the genotyped DNA samples were homozygous for the larger 4b allele and the remaining 57 samples (25%) were heterozygous (4b/4a). No DNA samples were homozygous for the shorter 4a allele with four 27 bp repeats. In case of ACE/ID, 47 (20%) of the DNA samples were homozygous for the insertion, 65 (28%) were homozygous for the deletion and the remaining 118 (51%) were heterozygous. The results obtained were verified by analyzing random amplicons using bi-directional sequencing and GeneScan 3.0 analyses with 100% concordance being observed. Using the microfluidic chip-based method, separation and DNA sizing and genotyping are rapidly accomplished. The DNA fragments are resolved clearly and the system allows quantitation. Finally, the microfluidic chip-based method may be used for both large- and small-scale genotyping studies.
Sohni
Youvraj R
YR
Translational Genomics Center, Mayo Clinic, Rochester, MN 55905, USA.
Burke
James P
JP
Dyck
Peter J
PJ
O'Kane
Dennis J
DJ
D000483:Alleles; D003327:Coronary Disease / Q000235:genetics; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics; D003928:Diabetic Nephropathies / Q000235:genetics; D017353:Gene Deletion; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D018895:Microsatellite Repeats; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA; D020080:Tandem Repeat Sequences
C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D007703:Peptidyl-Dipeptidase A