Use of an electrochemically labeled nucleotide terminator for known point mutation analysis.
A novel single nucleotide polymorphism (SNP) assay utilizing an electrochemically tagged chain terminator is described. The system employs the single-base extension (SBE) technique coupled to capillary gel electrophoresis with end-column electrochemical detection. A redox-labeled chain terminator, ferrocene-acycloATP, is used in the SBE reaction. When the mutation site corresponds to the labeled chain terminator, the extension product is rendered electroactive. The reaction mixture is subsequently separated by capillary gel electrophoresis and the extension product detected at the separation anode with sinusoidal voltammetry. This work demonstrates the first known SNP assay utilizing redox-active chain terminators coupled to electrochemical detection. The methodology presented could lead to a fast, simple, and cost-effective SNP scoring system.
Brazill
Sara
S
Department of Chemistry, Colgate University, Hamilton, NY 13346, USA. sbrazill@mail.colgate.edu
Hebert
Nicole E
NE
Kuhr
Werner G
WG
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D004563:Electrochemistry / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods; D005296:Ferrous Compounds; D006801:Humans; D000075163:Metallocenes; D015335:Molecular Probes; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide
D017931:DNA Primers; D005296:Ferrous Compounds; D000075163:Metallocenes; D015335:Molecular Probes; D000255:Adenosine Triphosphate; C004998:ferrocene