From gels to chips: "minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms.
In the minisequencing primer extension reaction, a DNA polymerase is used specifically to extend a primer that anneals immediately adjacent to the nucleotide position to be analyzed with a single labeled nucleoside triphospate complementary to the nucleotide at the variant site. The reaction allows highly specific detection of point mutations and single nucleotide polymorphisms (SNPs). Because all SNPs can be analyzed with high specificity at the same reaction conditions, minisequencing is a promising reaction principle for multiplex high-throughput genotyping assays. It is also a useful tool for accurate quantitative PCR-based analysis. This review discusses the different approaches, ranging from traditional gel-based formats to multiplex detection on microarrays that have been developed and applied to minisequencing assays.
Syvänen
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Department of Medical Sciences, Molecular Medicine, Uppsala University Hospital, Sweden. ann-christine.syvanen@medicin.uu.se
D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005838:Genotype; D006801:Humans; D009711:Nucleotides / Q000032:analysis; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D009711:Nucleotides; D004259:DNA-Directed DNA Polymerase