Towards automatic detection of point mutations: use of scintillating microplates in solid-phase minisequencing.
Simplification of molecular genetic techniques is one of the main features of large-scale clinical applications of mutation analysis. The solid-phase minisequencing method, which is based on single-nucleotide primer extension by a DNA polymerase on a solid support, is an easy way of detecting point mutations of previously known locations. Here the procedure was further simplified by the use of microplates made of scintillating plastics, a microplate format scintillation counter and an automatic microplate washer. DNA samples from patients with either a hereditary aspartylglucosaminidase (AGA) gene point mutation or an acquired N-ras gene mutation were analyzed by three different minisequencing detection procedures utilizing tritiated nucleotides. The new counting method with scintillating plates was compared to traditional liquid scintillation counting in scintillation vials or to another microplate format procedure, which requires addition of scintillation liquid. In all three methods, normal individuals, heterozygous carriers of the AGA mutation and homozygous patients could be unequivocally discriminated. The N-ras mutation in leukemic blasts could also be detected with high resolution. The coefficients of variation and reproducibility of the scintillating microplate method were almost identical to those of the traditional liquid scintillation assay, which was used as a reference method. The technical innovations adopted here for performing minisequencing assays reduce significantly the labor required without affecting the quality of the results.
Ihalainen
J
J
University of Helsinki, Finland.
Siitari
H
H
Laine
S
S
Syvänen
A C
AC
Palotie
A
A
D001227:Aspartylglucosylaminase / Q000235:genetics; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D008969:Molecular Sequence Data; D017354:Point Mutation
D001227:Aspartylglucosylaminase