Europium-labeled oligonucleotides to detect point mutations: application to PIZ alpha 1-antitrypsin deficiency.
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.
Dahlén
P
P
Wallac Oy, Turku, Finland.
Carlson
J
J
Liukkonen
L
L
Lilja
H
H
Siitari
H
H
Hurskainen
P
P
Iitä
A
A
Jeppsson
J O
JO
Lövgren
T
T
D001483:Base Sequence; D001710:Biotin; D015139:Blotting, Southern; D004247:DNA / Q000032:analysis; D005063:Europium; D005470:Fluorometry; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010641:Phenotype; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012996:Solutions; D013997:Time Factors; D000515:alpha 1-Antitrypsin / Q000235:genetics; D019896:alpha 1-Antitrypsin Deficiency
D015345:Oligonucleotide Probes; D012996:Solutions; D000515:alpha 1-Antitrypsin; D005063:Europium; D001710:Biotin; D004247:DNA