Microfluidic chip-based method for genotyping microsatellites, VNTRs and insertion/deletion polymorphisms

Youvraj R. Sohni, James P. Burke, Peter J Dyck, Dennis J. O'Kane

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)35-40
Number of pages6
JournalClinical Biochemistry
Volume36
Issue number1
DOIs
StatePublished - Jan 2003

Fingerprint

Minisatellite Repeats
Microfluidics
Polymorphism
Microsatellite Repeats
DNA
Medical problems
Nitric Oxide
Alleles
Genes
Linkage Disequilibrium
Diabetic Nephropathies
Nitric Oxide Synthase Type II
Peptidyl-Dipeptidase A
Diabetes Complications
Gene encoding
Oligonucleotide Array Sequence Analysis
Type 1 Diabetes Mellitus
Genetic Promoter Regions
Introns
Type 2 Diabetes Mellitus

Keywords

  • Genotyping
  • Microfluidic
  • Microsatellites
  • VNTRs

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Microfluidic chip-based method for genotyping microsatellites, VNTRs and insertion/deletion polymorphisms. / Sohni, Youvraj R.; Burke, James P.; Dyck, Peter J; O'Kane, Dennis J.

In: Clinical Biochemistry, Vol. 36, No. 1, 01.2003, p. 35-40.

Research output: Contribution to journalArticle

Sohni, Youvraj R. ; Burke, James P. ; Dyck, Peter J ; O'Kane, Dennis J. / Microfluidic chip-based method for genotyping microsatellites, VNTRs and insertion/deletion polymorphisms. In: Clinical Biochemistry. 2003 ; Vol. 36, No. 1. pp. 35-40.
@article{777c3c15ca2946de815923872e415e4a,
title = "Microfluidic chip-based method for genotyping microsatellites, VNTRs and insertion/deletion polymorphisms",
abstract = "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{\circledR} 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.",
keywords = "Genotyping, Microfluidic, Microsatellites, VNTRs",
author = "Sohni, {Youvraj R.} and Burke, {James P.} and Dyck, {Peter J} and O'Kane, {Dennis J.}",
year = "2003",
month = "1",
doi = "10.1016/S0009-9120(02)00420-4",
language = "English (US)",
volume = "36",
pages = "35--40",
journal = "Clinical Biochemistry",
issn = "0009-9120",
publisher = "Elsevier Inc.",
number = "1",

}

TY - JOUR

T1 - Microfluidic chip-based method for genotyping microsatellites, VNTRs and insertion/deletion polymorphisms

AU - Sohni, Youvraj R.

AU - Burke, James P.

AU - Dyck, Peter J

AU - O'Kane, Dennis J.

PY - 2003/1

Y1 - 2003/1

N2 - 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.

AB - 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.

KW - Genotyping

KW - Microfluidic

KW - Microsatellites

KW - VNTRs

UR - http://www.scopus.com/inward/record.url?scp=0037245580&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037245580&partnerID=8YFLogxK

U2 - 10.1016/S0009-9120(02)00420-4

DO - 10.1016/S0009-9120(02)00420-4

M3 - Article

C2 - 12554058

AN - SCOPUS:0037245580

VL - 36

SP - 35

EP - 40

JO - Clinical Biochemistry

JF - Clinical Biochemistry

SN - 0009-9120

IS - 1

ER -