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Clinical Chemistry 43: 1142-1150, 1997;
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(Clinical Chemistry. 1997;43:1142-1150.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Simple triple-label detection of seven cystic fibrosis mutations by time-resolved fluorometry

Pia Heinonen1,a, Antti Iitiä1,2, Toni Torresani3 and Timo Lövgren1

1 Department of Biotechnology, University of Turku, FIN-20520 Turku, Finland.

2 Current address: InnoTrac Diagnostics Oy, FIN-20520 Turku, Finland.

3 Division of Endocrinology, University Children's Hospital, CH-8032 Zürich, Switzerland.
a Author for correspondence. Fax 358-2-333 8050;

We describe a simple hybridization assay performed in microtitration wells with use of DNA probes labeled with three different lanthanide chelates for detection of seven mutations that cause cystic fibrosis. The assay is based on DNA amplification of four fragments containing the mutations ({Delta}F508, G1717->A, G542X, R553X, 3905 insertion T, W1282X, and N1303K) by PCR, followed by hybridization with short, allele-specific oligonucleotide probes labeled with europium, terbium, or samarium chelates. Because the technology makes it possible to hybridize three DNA probes simultaneously in one reaction, all 14 mutation-related alleles were detected in a total of five reaction wells. Blood spot specimens, obtained from children with cystic fibrosis, their parents, and their siblings, have been assayed, and for all the probes the positive signal-to-noise ratios are >10. Solution hybridization utilizing triple-label time-resolved fluorometry combined with PCR is a suitable procedure for large-scale screening and automation.


Key Words: indexing terms: allele-specific hybridization • PCR • hybridization assay • genetic disease • lanthanide chelate




The following articles in journals at HighWire Press have cited this article:


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A. Ylikoski, A. Elomaa, P. Ollikka, H. Hakala, V.-M. Mukkala, J. Hovinen, and I. Hemmila
Homogeneous Time-Resolved Fluorescence Quenching Assay (TruPoint) for Nucleic Acid Detection
Clin. Chem., October 1, 2004; 50(10): 1943 - 1947.
[Full Text] [PDF]

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 J. Clin. Endocrinol. Metab.Home page
M. Jiang, K. Aittomäki, C. Nilsson, P. Pakarinen, A. Iitiä, T. Torresani, H. Simonsen, V. Goh, K. Pettersson, A. de la Chapelle, et al.
The Frequency of an Inactivating Point Mutation (566C->T) of the Human Follicle-Stimulating Hormone Receptor Gene in Four Populations Using Allele-Specific Hybridization and Time-Resolved Fluorometry
J. Clin. Endocrinol. Metab., December 1, 1998; 83(12): 4338 - 4343.
[Abstract] [Full Text]


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