Determining pyrophosphate, useful e.g. for optimizing polymerase chain reaction conditions or nucleic acid synthesis, uses polymerase with pyrophosphorolysis activity and oligonucleotide as indicator

摘要

Determining pyrophosphate (PP) in reaction mixture and biological samples comprising using at least one polymerase (I) with pyrophosphorolysis activity and an indicator substrate (I) that generates a signal depending on the amount of PP present, is new. Determining pyrophosphate (PP) in reaction mixture and biological samples using at least one polymerase (I) with pyrophosphorolysis activity and an indicator substrate (I) that generates a signal depending on the amount of PP present. The new feature is that (I) is an at least partly double-stranded (ds) oligonucleotide (ON) of random sequence which is labeled at the 3'-end with a detectable functional group or molecule (A). In presence of (I), the labeled 3'-terminal nucleotide (nt), with attached (A), is released with consumption of a PP molecule, and this release is quantified to indicate the PP content. An Independent claim is also included for a reagent kit for optimization of PCR or other in vitro DNA/RNA synthesis reactions containing (I); polymerase; nucleotides; control template(s); instructions and optimized examples.