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

Autor:	P Dahlén, A Iitä, Hans Lilja, H Siitari, J Carlson, Timo Lövgren, J.-O. Jeppsson, Pertti Hurskainen, L Liukkonen
Rok vydání:	1993
Předmět:	Oligonucleotide Biochemistry (medical) Clinical Biochemistry chemistry.chemical_element Biology Molecular biology chemistry.chemical_compound Nucleic acid thermodynamics chemistry Fluorometer Biotinylation Nucleic acid Solution hybridization Europium DNA
Zdroj:	Clinical Chemistry. 39:1626-1631
ISSN:	1530-8561 0009-9147
DOI:	10.1093/clinchem/39.8.1626
Popis:	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.
Databáze:	OpenAIRE
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