Proofreading genotyping assays and electrochemical detection of SNPs

The use of proofreading DNA polymerases in genotyping assays offers the prospect of improved performance. To this end, we have recently used compatible DNA polymerases, protected primers, and substrates to implement proofreading single base extension (P-SBE) and proofreading allele-specific extension (PRASE) assays. Key aspects of the P-SBE and related proofreading assay formats are described here. For transduction of genotyping reactions into physical signals, electrochemical SBE implementations may offer simple, inexpensive assays in electrode array or electrophoretic formats. We have developed electrochemically-labeled nucleotides and electrode detection methods with a view to these applications. Detection of electrochemically-labeled SBE products on an oligonucleotide-modified gold electrode surface is demonstrated.