Nanopore sequencing using charge blockade labels

摘要

The invention relates to devices and methods for nanopore sequencing. The invention includes compositions and methods of nucleic acid sequencing using a single polymerase enzyme complex comprising a polymerase enzyme and a template nucleic acid attached proximal to a nanopore, and nucleotide analogs in solution comprising charge blockade label that are attached to the polyphosphate portion of the nucleotide analog such that the charge blockade labels are cleaved when the nucleotide analog is incorporated into a growing nucleic acid and the charge blockade label is detected by the nanopore to determine the presence and identity of the incorporated nucleotide and thereby determine the sequence of a template nucleic acid.

主权项

1. A method for determining sequence information about a template nucleic acid molecule comprising
providing a substrate having a nanopore, the nanopore having a top opening and a bottom opening, and having a single polymerase enzyme attached proximal to an opening, the polymerase enzyme complexed with a primed template nucleic acid; contacting the substrate with a sequencing reaction mixture comprising reagents required for polymerase mediated nucleic acid synthesis including two or more different types of nucleotide analogs, each of the types comprising a current blockade label attached to the phosphate portion of the type of nucleotide analogs through a linker, such that when polymerase mediated nucleic acid synthesis occurs the label is cleaved, and each of the types of nucleotide analogs having different current blockade characteristics, providing a voltage drop across the nanopore, whereby when the base portion of a nucleofide analog, which is a molecule corresponding to one of the two or more types of nucleotide analogs, is associated with the polymerase enzyme while it is being incorporated into a growing strand during nucleic acid synthesis, the current blockage label on the nucleotide analog at least partially enters the nanopore, resulting in a measureable change in current through the nanopore; measuring the current through the nanopore over time to detect the incorporation of nucleotide analogs into the growing strand; and using the measured current over time while the nucleotide analog is associated with the polymerase to detect and identify the incorporated nucleotide, thereby obtaining sequence information about the template nucleic acid.