Europium-labeled oligonucleotides to detect point mutations : Application to PI Z α1-antitrypsin deficiency
(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.
(Less)
- author
- Dahlen, P. ; Carlson, J. LU ; Liukkonen, L. ; Lilja, H. LU ; Siitari, H. ; Hurskainen, P. ; Iita, A. ; Jeppsson, J. O. LU and Lovgren, T.
- publishing date
- 1993
- 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
- American Association for Clinical Chemistry
- 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
- 2024-01-03 10:59:27
@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 = {{American Association for Clinical Chemistry}}, 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}}, }