SEQUENCING SYSTEMS WITH TWO SLOW-STEP POLYMERASE ENZYMES

摘要

Systems for nucleotide sequencing comprising producing polymerase reactions that exhibit two kinetically observable steps within an observable phase of the polymerase reaction. Two slow step systems can be produced, for example, by selecting the appropriate polymerase enzyme, polymerase reaction conditions including cofactors, and polymerase reaction substrates including the primed template and nucleotides.

主权项

1. A system for single-molecule nucleotide sequencing comprising:
a) a reaction mixture having: (i) a polymerase enzyme, (ii) polymerase reaction conditions including cofactors, and (iii) polymerase reaction substrates including a primed template and nucleotides comprising observable labels, such that a reaction comprising incorporation of the nucleotides into a growing nucleic acid occurs, wherein the polymerase enzyme, the polymerase reaction conditions, and the polymerase reaction substrates are selected such that the reaction exhibits two kinetically observable steps, each of which kinetically observable steps proceeds from an intermediate in which a nucleotide or a polyphosphate product is bound to the polymerase enzyme or each of which kinetically observable steps proceeds from an intermediate in which the nucleotide and the polyphosphate product are not bound to the polymerase enzyme; and (b) a detector to detect the observable label to measure the sequential incorporation of nucleotides into the growing nucleic acid thereby obtaining a sequence of the template