Patent ID: 8911972
Filing Date: 2014-12-16
Classification: C12Q

Abstract:
1. A method of sequencing comprising; providing a single polymerase enzyme complex comprising a polymerase enzyme, a nucleic acid template, and a primer; wherein the polymerase enzyme comprises a label which has a signal that changes when the enzyme undergoes a conformational change corresponding to translocation; contacting the complex with sequencing reagents including two or more types of nucleotides or nucleotide analogs, each labeled with a different label, whereby enzyme mediated addition of nucleotides or nucleotide analogs to the primer to produce a growing strand complementary to the template occurs, wherein the labels on each of the two or more types of nucleotides or nucleotide analogs, and the label on the polymerase enzyme each provide a distinct signal; monitoring in real time the distinct signals from the labels on each of the two or more types of nucleotides or nucleotide analogs, and the distinct signals from the label on the polymerase enzyme as the growing strand is produced; determining the type of nucleotide or nucleotide analog that has been incorporated by observing signals from the labeled nucleotides or nucleotide analogs during the enzyme mediated addition; determining whether a nucleotide or nucleotide analog is incorporated into the growing strand using observed signals from the labeled polymerase enzyme that are indicative of translocation; and combining the determination of the type of nucleotide or nucleotide analog with the determination of whether a nucleotide or nucleotide analog is incorporated, whereby observing both a signal from the label on a type of nucleotide or nucleotide analog and a signal from the label on the polymerase enzyme corresponding to translocation within the same time period indicates that a nucleotide or nucleotide analog of that type has been incorporated in that time period, and whereby observing only a signal from the label on a type of nucleotide or nucleotide analog, and no signal from the label on the polymerase enzyme corresponding to translocation within the same time period indicates that no nucleotide or nucleotide analog has been incorporated in that time period, to obtain more accurate sequencing than without the determination of whether a nucleotide or nucleotide analog has been incorporated, whereby including the signals from the labeled polymerase reduces errors due to branching.