Polymerase compositions, methods of making and using same
US-10240134-B2 · Mar 26, 2019 · US
Method of nucleic acid sequence determination
US11976323B2 · US · B2
| Field | Value |
|---|---|
| Publication number | US-11976323-B2 |
| Application number | US-202016736071-A |
| Country | US |
| Kind code | B2 |
| Filing date | Jan 7, 2020 |
| Priority date | Apr 29, 2016 |
| Publication date | May 7, 2024 |
| Grant date | May 7, 2024 |
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- Title
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Abstract
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Provided are sequencing-by-binding methods of detecting cognate nucleotides using a crippled DNA polymerizing enzyme that possesses the ability to bind the next correct nucleotide downstream of a primer in a template-dependent fashion, but does not possess the activity needed to promote phosphodiester bond formation. Use of the crippled DNA polymerase permits interrogation of one nucleotide at a time, without incorporation of any nucleotide. Labeled nucleotides, such as fluorescently labeled nucleotides, can be used in conjunction with the crippled DNA polymerase to establish cognate nucleotide identity in a rapid manner.
First claim
Opening claim text (preview).
What is claimed is: 1. A mutant DNA polymerase comprising a polypeptide sequence, said polypeptide sequence comprising SEQ ID NO:12, wherein said mutant DNA polymerase forms ternary complexes with primed template nucleic acid molecules and cognate nucleotides, and wherein said mutant DNA polymerase is incapable of magnesium-catalyzed phosphodiester bond formation. 2. The mutant DNA polymerase of claim 1 , wherein the mutant DNA polymerase catalyzes formation of phosphodiester bonds in the presence of manganese ions. 3. The mutant DNA polymerase of claim 1 , wherein the polypeptide sequence of the mutant DNA polymerase comprises SEQ ID NO:1 with the exception of comprising SEQ ID NO:12 and having glutamate at amino acid position 381. 4. The mutant DNA polymerase of claim 1 , wherein the polypeptide sequence of the mutant DNA polymerase comprises SEQ ID NO:2 with the exception of comprising SEQ ID NO:12 and having glutamate at amino acid position 364. 5. The mutant DNA polymerase of claim 1 , wherein the polypeptide sequence of the mutant DNA polymerase comprises SEQ ID NO:3 with the exception of comprising SEQ ID NO:12 and having glutamate at amino acid position 355. 6. The mutant DNA polymerase of claim 1 , further comprising an exogenous label. 7. A ternary complex, comprising the mutant DNA polymerase of claim 1 , a primed template nucleic acid and a next correct nucleotide for the primed template nucleic acid. 8. The ternary complex of claim 7 , wherein the next correct nucleotide comprises an exogenous label. 9. The ternary complex of claim 7 , wherein the mutant DNA polymerase comprises an exogenous label.
Assignees
Inventors
Classifications
- C12Q1/6869Primary
Methods for sequencing · CPC title
DNA-directed RNA polymerase (2.7.7.6) · CPC title
involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title
DNA-directed RNA polymerase (2.7.7.6) · CPC title
DNA polymerase · CPC title
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- What does patent US11976323B2 cover?
- Provided are sequencing-by-binding methods of detecting cognate nucleotides using a crippled DNA polymerizing enzyme that possesses the ability to bind the next correct nucleotide downstream of a primer in a template-dependent fashion, but does not possess the activity needed to promote phosphodiester bond formation. Use of the crippled DNA polymerase permits interrogation of one nucleotide at …
- Who is the assignee on this patent?
- Omniome Inc, Pacific Biosciences California Inc
- What technology area does this patent fall under?
- Primary CPC classification C12Q1/6869. Mapped technology areas include Chemistry & Metallurgy.
- When was this patent published?
- Publication date Tue May 07 2024 00:00:00 GMT+0000 (Coordinated Universal Time) (B2). Legal status and post-grant events are not shown on this page.
- What related patents are in patentsdb?
- We list 8 related publications on this page (citations in our corpus or others sharing the same primary CPC).