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Modified nucleotide reagents

A nucleotide and compound technology, used in biological testing, microbial determination/inspection, fluorescence/phosphorescence, etc.

Active Publication Date: 2018-09-28
PACIFIC BIOSCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In turn, lower precision may require multiple coverage to identify a specific sequence with a high level of confidence

Method used

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Examples

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Embodiment 1

[0406] Embodiment 1. Synthesis of double biotin nucleotide compounds

[0407] Various nucleotide compounds containing dual biotin linkers have been synthesized for use in single-molecule real-time sequencing reactions. Avidin proteins have been used to assemble these compounds with dye-labeled compounds, or intermediate forms thereof, also comprising a dual-biotin linker, resulting in dye-labeled nucleotide analog complexes, e.g. as described in Example 2 stated and as Figure 7A to Figure 7D and Figure 7F shown. Additional examples of labeled nucleotide analogs that have been prepared according to these methods are shown schematically in Figure 3E to Figure 3O '. Many analogs exhibit improved photostability, brightness, and reaction kinetics in automated DNA sequencing reactions involving DNA polymerases. See also US Patent Application Publication No. 2013 / 0316912 Al. Example 3 describes the use of assembled fluorescent nucleotide reagent complexes in real-time sequ...

Embodiment 2

[0444] Example 2. Assembly of dye-labeled nucleotide analogs

[0445] The mononucleotide and dinucleotide compounds described above are assembled into dye-labeled Nucleotide analogs. For most of the kinetic experiments described in Example 3, a single avidin protein and a protein such as Figure 4A Simple, unshielded dye-labeled compounds, such as the dye-labeled compounds shown in Figure 1, can be assembled into nucleotide compounds. Such assembly can be performed as described in US Patent Application Publication No. 2013 / 0316912 Al. For example using Figure 7A to Figure 7D and Figure 7F The pathway shown, also assembles more complex analog structures. As described in Example 3, these analogs (e.g. Figure 19A Analogs depicted in ) were evaluated.

Embodiment 3

[0446] Example 3. Use of dye-labeled nucleotide analogs in real-time sequencing reactions

[0447] Single-molecule real-time sequencing reactions using the fluorescent nucleotide analogs described in Example 2 were performed in a zero-mode waveguide ("ZMW") array with 3000 discrete cores. Reactions were visualized using a highly multiplexed confocal fluorescence microscope that provided a directional distribution of illumination, eg, each core as an independent spot. See, eg, US Patent No. 7,714,303, which is hereby incorporated by reference in its entirety for all purposes. Fluorescent signals from various ZMWs were detected using an EMCCD camera, and the signals were subjected to pulse recognition and base calling processes. See, eg, US Patent No. 8,182,993, which is hereby incorporated by reference in its entirety for all purposes. Sequencing was generally performed as described in Eid, J et al. Science 323:133-138 and the corresponding supplementary information include...

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Abstract

Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful inthe preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 62 / 258,414, filed November 20, 2015, the disclosure of which is incorporated herein by reference in its entirety. [0003] sequence listing [0004] This application includes the sequence listing shown in an ASCII-compatible text file named "1407-00-012WO1_2016-11-21_Seq_list_ST25.txt" created on November 21, 2016, which contains 136,044 bytes and will be published in full Incorporated herein by reference. Background technique [0005] The development of novel modified nucleotide reagents, especially those containing fluorescent labels, has increased the performance of nucleotide sequencing reactions, such as providing nucleotides that identify all four bases in a single reaction solution Sequencing reactions. This approach has been used for "real-time" detection of incorporation events, where the act of incorporation produces a signal event that ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09G01N21/64G01N33/53
CPCC07H19/10C07H19/207C09B69/105C12Q1/6869C12Q1/6876
Inventor 吉恩·沈史帝芬·余卢博米尔·塞博路易斯·布罗格利
Owner PACIFIC BIOSCIENCES
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