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Clinical Chemistry 39: 1626-1631, 1993;
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Clinical Chemistry, Vol 39, 1626-1631, Copyright © 1993 by American Association for Clinical Chemistry

Europium-labeled oligonucleotides to detect point mutations: application to PIZ alpha 1-antitrypsin deficiency

P Dahlen, J Carlson, L Liukkonen, H Lilja, H Siitari, P Hurskainen, A Iita, JO Jeppsson and T Lovgren
Wallac Oy, Turku, Finland.

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 alpha 1-antitrypsin gene. Evaluation of whole- blood samples spotted on Guthrie cards demonstrated successful accuracy of the method.


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