US-12624390-B2 - Distinguishing sequences by detecting polymerase dissociation

Abstract

A method for determining the presence of an allele, including (a) binding a polymerase to a double stranded nucleic acid that includes a primer hybridized to a template, the template including a first allele of a locus; (b) adding a nucleotide to the primer via catalytic activity of the polymerase, thereby producing an extended nucleic acid; (c) dissociating the polymerase from the extended nucleic acid; (d) detecting dissociation of the polymerase from the extended nucleic acid; and (e) comparing the dissociation of the polymerase from the extended nucleic acid to dissociation of the polymerase from a second double stranded nucleic acid, the second double stranded nucleic acid including a primer hybridized to the same position of the locus as the primer of the extended nucleic acid.

Inventors

• Denis Malyshev
• Sean Stromberg

Assignees

• PACIFIC BIOSCIENCES OF CALIFORNIA, INC.

Dates

Publication Date

20260512

Application Date

20220927

Claims (1)

1. 1 . A method for determining the presence of an allele, comprising (a) binding a polymerase to a double stranded nucleic acid that comprises an extendible primer hybridized to a template; (b) adding a plurality of nucleotides to the extendable primer via catalytic activity of the polymerase, thereby producing an extended primer that is complementary to a homopolymer region of the allele; (c) dissociating the polymerase from the extended double stranded nucleic acid; and (d) detecting dissociation of the polymerase from the extended double stranded nucleic acid; (e) comparing the dissociation of the polymerase from the extended double stranded nucleic acid to dissociation of the polymerase from a second extended double stranded nucleic acid, thereby determining the allele at or near the homopolymer region, wherein the allele is identified based on the comparing of the dissociations in step (e); wherein step (b) comprises a step in a sequencing method; and wherein the polymerase comprises an exogenous label.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation of U.S. application Ser. No. 16/486,062, filed Aug. 14, 2019, which is the National Stage Application of International Application No. PCT/US2018/018118, filed Feb. 14, 2018, which claims priority to U.S. Provisional Application No. 62/459,437, filed Feb. 15, 2017, which are incorporated by reference herein in their entirety. SEQUENCE LISTING A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. The Sequence Listing has been filed as an electronic document via EFS-Web in XML code. The electronic document, created on Sep. 23, 2022, is entitled “097128-1348887-034US2_ST26.xml”, and is 8,651 bytes in size. BACKGROUND The present disclosure relates generally to molecular diagnostics, and has specific applicability to evaluation of nucleic acids. Small differences in nucleic acid sequences can result in significant differences in biological function. For example, single nucleotide polymorphisms (SNPs) in the human genome underlie differences in susceptibility to disease. A wide range of human diseases, such as sickle-cell anemia, β-thalassemia, Alzheimer's Disease and cystic fibrosis result from SNPs. Recent advances in genotyping and DNA sequencing have identified many SNPs that are associated with the probability of developing a variety of diseases and conditions. Such SNPs can be useful for diagnosis and prognosis of the disease or conditions to which they have been associated. Furthermore, many of these SNPs are likely to be therapeutically relevant genetic variants and/or involved in genetic predisposition to disease. However, accurate diagnostic correlations generally require evaluation of large SNP panels (e.g. on a genome-wide scale) for a large population of individuals. Currently available methods are costly and time consuming, which is unfavorable for scaling the methods to clinically meaningful levels. Thus, there exists a need for efficient methods to detect a large variety of SNPs, or other nucleic acid polymorphisms, often in many individuals. The present disclosure satisfies this need and provides related advantages as well. BRIEF SUMMARY The present disclosure provides a method for determining the presence of a nucleic acid allele. The method can include the steps of (a) binding a polymerase to a double stranded nucleic acid that includes a primer hybridized to a template, the template including a first allele of a locus; (b) adding a nucleotide to the primer via catalytic activity of the polymerase, thereby producing an extended double stranded nucleic acid; (c) dissociating the polymerase from the extended double stranded nucleic acid; (d) detecting dissociation of the polymerase from the extended double stranded nucleic acid; and (e) comparing the dissociation of the polymerase from the extended double stranded nucleic acid to dissociation of the polymerase from a second double stranded nucleic acid, the second double stranded nucleic acid including a primer hybridized to the same position of the locus as the primer of the extended double stranded nucleic acid, thereby determining the presence of the first allele in the template nucleic acid. Additionally, a method is provided for distinguishing nucleic acid alleles by carrying out the steps of (a) providing a first template nucleic acid including a first allele of a locus and a second template nucleic acid including a second allele of the locus, wherein the first and second templates are hybridized to a primer, thereby providing first and second double stranded nucleic acids, respectively; (b) binding polymerases to the first and second double stranded nucleic acids; (c) adding nucleotides to the primers of the first and second double stranded nucleic acids via catalytic activity of the polymerases, thereby producing an extended first double stranded nucleic acid and an extended second double stranded nucleic acid; (d) dissociating the polymerases from the extended double stranded nucleic acids; (e) detecting dissociation of the polymerase from the extended double stranded nucleic acids; and (f) determining a difference in the dissociation of the polymerase from the first extended double stranded nucleic acid to the dissociation of the polymerase from the second extended double stranded nucleic acid, thereby distinguishing the first allele from the second allele. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a graph showing a plot of initial dissociation rates measured during incorporation/dissociation steps for each cycle of a Sequencing By Binding' run performed with a PhiX wild type template and the PhiX m2 mutant. FIG. 1B is a graph showing a plot of initial dissociation rates measured during incorporation/dissociation steps for each cycle of a Sequencing By Binding' run performed with a PhiX wild type template and the PhiX m3 mutant. DETAILED DESCRIPTION The present disclosure provides methods for detecting or identi