Colorimetric solid-phase minisequencing assay illustrated by detection of alpha 1-antitrypsin Z mutation.
In solid-phase minisequencing, a defined point mutation is detected in microtiter plate-immobilized DNA by a single nucleotide primer extension reaction. We have here developed the method into a colorimetric assay and applied it to the detection of the Z mutation of the alpha 1-antitrypsin gene. We used novel nucleoside triphosphates modified with dinitrophenyl (DNP) hapten, permitting detection by anti-DNP-alkaline phosphatase conjugate, with p-nitrophenyl phosphate as substrate. The Z mutation is detected in two reactions: DNP-labeled dCTP is incorporated when the template is normal, DNP-dUTP when the Z mutation is present. Both modified nucleotides were incorporated with high specificity and with an efficiency similar to that of unmodified nucleotides. The test results are measured by spectrophotometry, yielding quantitative absorbance values. Calculation of the ratio of C to U signal permitted unambiguous distinction of normal homozygous, ZZ homozygous, and ZM heterozygous genotypes. The colorimetric minisequencing assay is rapid, standardized, and automatable, and thus provides an accurate and simple alternative for the analysis of known point mutations.
Harju
L
L
Orion Corp., Orion Pharmaceutica, Biotechnology, Valimotie, Helsinki, Finland.
Weber
T
T
Alexandrova
L
L
Lukin
M
M
Ranki
M
M
Jalanko
A
A
D001483:Base Sequence; D003124:Colorimetry / Q000379:methods* / Q000706:statistics & numerical data; D004247:DNA / Q000737:chemistry; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004140:Dinitrophenols; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D000515:alpha 1-Antitrypsin / Q000235:genetics*
D003845:Deoxycytosine Nucleotides; D003856:Deoxyuracil Nucleotides; D004140:Dinitrophenols; D000515:alpha 1-Antitrypsin; C027078:deoxyuridine triphosphate; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA