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Europium-labeled oligonucleotides to detect point mutations : Application to PI Z α1-antitrypsin deficiency

Dahlen, P. ; Carlson, J. LU ; Liukkonen, L. ; Lilja, H. LU orcid ; Siitari, H. ; Hurskainen, P. ; Iita, A. ; Jeppsson, J. O. LU and Lovgren, T. (1993) In Clinical Chemistry 39(8). p.1626-1631
Abstract

We describe a novel assay for detection of point mutations. The method combines the specificity and sensitivity of the polymerase chain reaction (PCR) and allele-specific oligonucleotides (ASO) with highly sensitive time- resolved fluorometry. ASO probes differing by a single base substitution and labeled with europium (Eu) chelates were hybridized in solution simultaneously with a biotinylated oligomer to a PCR-amplified nucleic acid fragment. The hybrids formed were then collected onto streptavidin-coated microtitration wells. Subsequently, the hybrids were washed under stringent conditions and the remaining ASO probe was measured in a time-resolved fluorometer. We discuss the strategy underlying the design of the Eu-labeled ASO... (More)

We describe a novel assay for detection of point mutations. The method combines the specificity and sensitivity of the polymerase chain reaction (PCR) and allele-specific oligonucleotides (ASO) with highly sensitive time- resolved fluorometry. ASO probes differing by a single base substitution and labeled with europium (Eu) chelates were hybridized in solution simultaneously with a biotinylated oligomer to a PCR-amplified nucleic acid fragment. The hybrids formed were then collected onto streptavidin-coated microtitration wells. Subsequently, the hybrids were washed under stringent conditions and the remaining ASO probe was measured in a time-resolved fluorometer. We discuss the strategy underlying the design of the Eu-labeled ASO probes for the solution hybridization assay. The method was applied to the detection of the Z-mutation in the α1-antitrypsin gene. Evaluation of whole-blood samples spotted on Guthrie cards demonstrated successful accuracy of the method.

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author
; ; ; ; ; ; ; and
publishing date
type
Contribution to journal
publication status
published
subject
keywords
allele- specific oligonucleotides, biotin-streptavidin interaction, fluorometry, heritable disorders, polymerase chain reaction
in
Clinical Chemistry
volume
39
issue
8
pages
6 pages
publisher
Oxford University Press
external identifiers
  • pmid:8353948
  • scopus:0027177978
ISSN
0009-9147
DOI
10.1093/clinchem/39.8.1626
language
English
LU publication?
no
id
78feab12-ffb6-4983-92ae-f8e57636d691
date added to LUP
2022-12-17 17:05:19
date last changed
2025-10-14 13:02:34
@article{78feab12-ffb6-4983-92ae-f8e57636d691,
  abstract     = {{<p>We describe a novel assay for detection of point mutations. The method combines the specificity and sensitivity of the polymerase chain reaction (PCR) and allele-specific oligonucleotides (ASO) with highly sensitive time- resolved fluorometry. ASO probes differing by a single base substitution and labeled with europium (Eu) chelates were hybridized in solution simultaneously with a biotinylated oligomer to a PCR-amplified nucleic acid fragment. The hybrids formed were then collected onto streptavidin-coated microtitration wells. Subsequently, the hybrids were washed under stringent conditions and the remaining ASO probe was measured in a time-resolved fluorometer. We discuss the strategy underlying the design of the Eu-labeled ASO probes for the solution hybridization assay. The method was applied to the detection of the Z-mutation in the α<sub>1</sub>-antitrypsin gene. Evaluation of whole-blood samples spotted on Guthrie cards demonstrated successful accuracy of the method.</p>}},
  author       = {{Dahlen, P. and Carlson, J. and Liukkonen, L. and Lilja, H. and Siitari, H. and Hurskainen, P. and Iita, A. and Jeppsson, J. O. and Lovgren, T.}},
  issn         = {{0009-9147}},
  keywords     = {{allele- specific oligonucleotides; biotin-streptavidin interaction; fluorometry; heritable disorders; polymerase chain reaction}},
  language     = {{eng}},
  number       = {{8}},
  pages        = {{1626--1631}},
  publisher    = {{Oxford University Press}},
  series       = {{Clinical Chemistry}},
  title        = {{Europium-labeled oligonucleotides to detect point mutations : Application to PI Z α<sub>1</sub>-antitrypsin deficiency}},
  url          = {{http://dx.doi.org/10.1093/clinchem/39.8.1626}},
  doi          = {{10.1093/clinchem/39.8.1626}},
  volume       = {{39}},
  year         = {{1993}},
}