| 摘要 |
The invention herein disclosed provides for devices, reagents, and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore. Of particular note is the use of reagents to rapidly sequence a polynucleotide. The invention is of particular use in the fields of forensic biology, molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof. |
| 主权项 |
1. A reagent for determining the nucleotide sequence of a target polynucleotide in a sample, the reagent comprising a first reagent component, a second reagent component, a third reagent component, and a DNA modifying enzyme, (i) wherein the first reagent component comprises a 1st polynucleotide partial duplex, the 1st polynucleotide partial duplex comprising a polynucleotide duplex and a first polynucleotide single strand and a second polynucleotide single strand, the polynucleotide duplex comprising a blocking oligomer and a loading oligomer, wherein a portion of the blocking oligomer comprises a nucleotide sequence that is the complement of a portion of the loading oligomer, wherein a portion of the blocking oligomer comprises a nucleotide sequence that is not the complement of a portion of the loading oligomer, wherein the blocking oligomer is annealed to the loading oligomer between the complementary portion of the blocking oligomer and the complementary portion of the loading oligomer thereby creating a proximal portion of the polynucleotide partial duplex, whereby the portion of the blocking oligomer that is not the complement of the loading oligomer is the first single strand and the portion of the loading oligomer that is not the complement of the blocking oligomer is the second single strand, and wherein the blocking oligomer first single strand and the loading oligomer second single strand comprise a distal portion of the polynucleotide partial duplex; (ii) wherein the second reagent component comprises a 2nd polynucleotide partial duplex, the 2nd polynucleotide partial duplex comprising a single polynucleotide, wherein a first portion of the single polynucleotide is substantially the complement of a second portion of the polynucleotide, and wherein the 2nd polynucleotide partial duplex comprises the first portion of the single polynucleotide is annealed to the second portion of the polynucleotide thereby creating a hairpin structure, the hairpin structure comprising a hairpin loop, a hairpin stem, the hairpin stem further comprising at least one acridine nucleotide residue, and third portion of the polynucleotide that is not annealed to either portion of the polynucleotide, the third portion further comprising a restriction endonuclease site; (iii) wherein the third reagent component comprises the target polynucleotide, the target polynucleotide substantially comprising a double strand polynucleotide and a first end and a second end, wherein the first end comprises a first strand that is the complement of the blocking oligomer and a second strand that is the complement of the loading oligomer, wherein the second end comprises a polynucleotide sequence that is the complement of the hairpin structure; and wherein the first and second and third reagent components are at an equimolar ratio. |
