Minisequencing with acyclonucleoside triphosphates tethered to lanthanide(III) chelates.
Four acyclic nucleoside triphosphates (derivatives of cytosine, thymine, 7-deazaadenine, and 7-deazaguanine) labeled with nonluminescent europium, terbium, dysprosium, and samarium chelates of 2,2',2'',2'''-[[4-(4-isothiocyanatophenyl)ethyl]pyridine-2,6-diyl]bis(methylenenitrilo)]tetrakis(acetic acid) were applied to minisequencing using two mutations (Delta F 508 and 1717-1 G to A) of cystic fibrosis as a model system. When synthetic targets were used, all four alleles involved could be analyzed in a single reaction using four terminating substrates labeled with four different lanthanide(III) chelates and DELFIA technology for detection. Blood spot samples without DNA isolations were used for PCR amplification and genotyping the target mutations by minisequencing. The single- and dual-labeled minisequencing assays were robust, while the four-label assay still requires further optimization of the multiplexed PCR amplification.
Ollikka
Pia
P
PerkinElmer Life and Analytical Sciences, Turku Site, POB 10, FI-20101 Turku, Finland.
Ylikoski
Alice
A
Kaatrasalo
Annukka
A
Harvala
Heli
H
Hakala
Harri
H
Hovinen
Jari
J
D001483:Base Sequence; D002614:Chelating Agents / Q000737:chemistry*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D005838:Genotype; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry* / Q000235:genetics*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013194:Staining and Labeling
D002614:Chelating Agents; D028581:Lanthanoid Series Elements; D009711:Nucleotides; D009841:Oligonucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator