Pathogen analysis and genetic predisposition testing using microelectronic arrays and isothermal amplification

C. F. Edman, P. Mehta, Richard Press, C. A. Spargo, G. T. Walker, M. Nerenberg

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

Background: A simple yet powerful tool for providing for rapid gene identification in the clinic would be the combination of isothermal gene amplification with electronic microchip analysis. This is a first report of such a union of these technologies. Methods: The first assay demonstrates discrimination between four bacterial pathogens. For this, one portion of the bacterial 16S rRNA gene encompassing a microheterogeneous region was isothermally amplified using Strand Displacement Amplification (SDA). Type identification was then made by 'sandwich' assay format either using selective electronic hybridization of amplicons to sequence-specific capture oligonucleotides and a universal, fluorescently labeled reporter oligonucleotide, or, alternatively, sequence-specific reporters and a universal capture oligonucleotide. The second assay tested for the presence or absence of the Factor V Leiden point mutation using DNA obtained from 18 patients in a blind assay. For this, allele-specific SDA was developed. Following amplification using a sense-biotinylated primer and either the corresponding antisense wild type or mutant primer, multiple patient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide. Positive signals were scored as greater than or equal to two times the background. Results: Bacterial type-specific signals were between 3- to 10-fold greater than nonspecific in both assay formats. Using allele-specific SDA, 100% agreement was observed between PAGE analysis, microarray results, and clinical diagnosis in Factor V mutation analysis. Conclusions: We demonstrated two model clinical assays combining amplified materials and microelectronic arrays, one potentially suitable for pathogen screening and the other for a deleterious genetic mutation.

Original languageEnglish (US)
Pages (from-to)93-101
Number of pages9
JournalJournal of Investigative Medicine
Volume48
Issue number2
StatePublished - 2000
Externally publishedYes

Fingerprint

Genetic Testing
Pathogens
Microelectronics
Oligonucleotides
Amplification
Assays
Testing
Microchip Analytical Procedures
Alleles
Microarrays
Mutation
Factor V
Gene Amplification
Microarray Analysis
Genes
rRNA Genes
Point Mutation
Technology
DNA
Screening

Keywords

  • Bacteria
  • DNA
  • Factor V
  • Fluorescent
  • Hybridization

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Pathogen analysis and genetic predisposition testing using microelectronic arrays and isothermal amplification. / Edman, C. F.; Mehta, P.; Press, Richard; Spargo, C. A.; Walker, G. T.; Nerenberg, M.

In: Journal of Investigative Medicine, Vol. 48, No. 2, 2000, p. 93-101.

Research output: Contribution to journalArticle

Edman, C. F. ; Mehta, P. ; Press, Richard ; Spargo, C. A. ; Walker, G. T. ; Nerenberg, M. / Pathogen analysis and genetic predisposition testing using microelectronic arrays and isothermal amplification. In: Journal of Investigative Medicine. 2000 ; Vol. 48, No. 2. pp. 93-101.
@article{e080017b6f834114875a15add527085d,
title = "Pathogen analysis and genetic predisposition testing using microelectronic arrays and isothermal amplification",
abstract = "Background: A simple yet powerful tool for providing for rapid gene identification in the clinic would be the combination of isothermal gene amplification with electronic microchip analysis. This is a first report of such a union of these technologies. Methods: The first assay demonstrates discrimination between four bacterial pathogens. For this, one portion of the bacterial 16S rRNA gene encompassing a microheterogeneous region was isothermally amplified using Strand Displacement Amplification (SDA). Type identification was then made by 'sandwich' assay format either using selective electronic hybridization of amplicons to sequence-specific capture oligonucleotides and a universal, fluorescently labeled reporter oligonucleotide, or, alternatively, sequence-specific reporters and a universal capture oligonucleotide. The second assay tested for the presence or absence of the Factor V Leiden point mutation using DNA obtained from 18 patients in a blind assay. For this, allele-specific SDA was developed. Following amplification using a sense-biotinylated primer and either the corresponding antisense wild type or mutant primer, multiple patient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide. Positive signals were scored as greater than or equal to two times the background. Results: Bacterial type-specific signals were between 3- to 10-fold greater than nonspecific in both assay formats. Using allele-specific SDA, 100{\%} agreement was observed between PAGE analysis, microarray results, and clinical diagnosis in Factor V mutation analysis. Conclusions: We demonstrated two model clinical assays combining amplified materials and microelectronic arrays, one potentially suitable for pathogen screening and the other for a deleterious genetic mutation.",
keywords = "Bacteria, DNA, Factor V, Fluorescent, Hybridization",
author = "Edman, {C. F.} and P. Mehta and Richard Press and Spargo, {C. A.} and Walker, {G. T.} and M. Nerenberg",
year = "2000",
language = "English (US)",
volume = "48",
pages = "93--101",
journal = "Journal of Investigative Medicine",
issn = "1081-5589",
publisher = "Lippincott Williams and Wilkins",
number = "2",

}

TY - JOUR

T1 - Pathogen analysis and genetic predisposition testing using microelectronic arrays and isothermal amplification

AU - Edman, C. F.

AU - Mehta, P.

AU - Press, Richard

AU - Spargo, C. A.

AU - Walker, G. T.

AU - Nerenberg, M.

PY - 2000

Y1 - 2000

N2 - Background: A simple yet powerful tool for providing for rapid gene identification in the clinic would be the combination of isothermal gene amplification with electronic microchip analysis. This is a first report of such a union of these technologies. Methods: The first assay demonstrates discrimination between four bacterial pathogens. For this, one portion of the bacterial 16S rRNA gene encompassing a microheterogeneous region was isothermally amplified using Strand Displacement Amplification (SDA). Type identification was then made by 'sandwich' assay format either using selective electronic hybridization of amplicons to sequence-specific capture oligonucleotides and a universal, fluorescently labeled reporter oligonucleotide, or, alternatively, sequence-specific reporters and a universal capture oligonucleotide. The second assay tested for the presence or absence of the Factor V Leiden point mutation using DNA obtained from 18 patients in a blind assay. For this, allele-specific SDA was developed. Following amplification using a sense-biotinylated primer and either the corresponding antisense wild type or mutant primer, multiple patient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide. Positive signals were scored as greater than or equal to two times the background. Results: Bacterial type-specific signals were between 3- to 10-fold greater than nonspecific in both assay formats. Using allele-specific SDA, 100% agreement was observed between PAGE analysis, microarray results, and clinical diagnosis in Factor V mutation analysis. Conclusions: We demonstrated two model clinical assays combining amplified materials and microelectronic arrays, one potentially suitable for pathogen screening and the other for a deleterious genetic mutation.

AB - Background: A simple yet powerful tool for providing for rapid gene identification in the clinic would be the combination of isothermal gene amplification with electronic microchip analysis. This is a first report of such a union of these technologies. Methods: The first assay demonstrates discrimination between four bacterial pathogens. For this, one portion of the bacterial 16S rRNA gene encompassing a microheterogeneous region was isothermally amplified using Strand Displacement Amplification (SDA). Type identification was then made by 'sandwich' assay format either using selective electronic hybridization of amplicons to sequence-specific capture oligonucleotides and a universal, fluorescently labeled reporter oligonucleotide, or, alternatively, sequence-specific reporters and a universal capture oligonucleotide. The second assay tested for the presence or absence of the Factor V Leiden point mutation using DNA obtained from 18 patients in a blind assay. For this, allele-specific SDA was developed. Following amplification using a sense-biotinylated primer and either the corresponding antisense wild type or mutant primer, multiple patient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide. Positive signals were scored as greater than or equal to two times the background. Results: Bacterial type-specific signals were between 3- to 10-fold greater than nonspecific in both assay formats. Using allele-specific SDA, 100% agreement was observed between PAGE analysis, microarray results, and clinical diagnosis in Factor V mutation analysis. Conclusions: We demonstrated two model clinical assays combining amplified materials and microelectronic arrays, one potentially suitable for pathogen screening and the other for a deleterious genetic mutation.

KW - Bacteria

KW - DNA

KW - Factor V

KW - Fluorescent

KW - Hybridization

UR - http://www.scopus.com/inward/record.url?scp=0033780280&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033780280&partnerID=8YFLogxK

M3 - Article

C2 - 10736968

AN - SCOPUS:0033780280

VL - 48

SP - 93

EP - 101

JO - Journal of Investigative Medicine

JF - Journal of Investigative Medicine

SN - 1081-5589

IS - 2

ER -