Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay.
A solid phase DNA sequencing method for non-radioactive detection of single base changes without the need for electrophoresis is presented. The concept relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (P. Nyrén, 1987, Anal. Biochem. 167, 235-238). Immobilized DNA fragments amplified with the polymerase chain reaction are used as template. A detection primer is annealed in front of the mutation and four aliquots of this mixture are incubated with DNA polymerase and one of the four different dideoxynucleotides. The presence or absence of an incorporated dideoxynucleotide is thereafter monitored by the release of inorganic pyrophosphate during the following primer extension step. We show that the concept can be used for sequencing of single bases as well as stepwise analysis of several subsequent bases.
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
P
P
Nyrén
B
B
Pettersson
M
M
Uhlén
D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase; D011756:Diphosphates; D005069:Evaluation Studies as Topic; D015751:Genes, pol; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000706:statistics & numerical data
D004279:DNA, Viral; D011756:Diphosphates; D004259:DNA-Directed DNA Polymerase