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Method of preparation of nanopore and uses thereof

A nano-scale, porous technology, used in the preparation of nano-pores and their uses, can solve problems such as expensive

Active Publication Date: 2015-02-25
THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the methods are expensive and may not provide sequence information for a period of time and with the level of precision that may be necessary to diagnose and / or treat a subject

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  • Method of preparation of nanopore and uses thereof
  • Method of preparation of nanopore and uses thereof
  • Method of preparation of nanopore and uses thereof

Examples

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example 1

[0734] I. Design and Synthesis of Modified Nucleotides

[0735] Using various phosphate-linked nucleotides with tags of different sizes or groups attached to the terminal phosphates of the nucleotides, the effect of the bulkiness of the tagged polyphosphate on the electronic blocking signal generated by the nanopore was determined role. The structures of four phosphate-tagged nucleoside-5'-polyphosphates are shown in Figure 23 middle. First, a series of nucleoside-5'-triphosphates, tetraphosphates, pentaphosphates and hexaphosphates are synthesized. In these nucleotides, a terminal phosphate is attached to a linker through which a different tag (such as ethylene glycol of different length and mass or other molecule that increases the bulkiness or charge of the released polyphosphate) passes connect. These nucleotides were tested through the nanopore to determine which tags or bulky groups attached to the terminal phosphates were associated with more pronounced differ...

example 2

[0778] I. Synthesis of PEG-labeled deoxyguanosine-5'-tetraphosphate (dG4P-PEG):

[0779] according to Figure 38 Synthesis of PEG-labeled deoxyguanosine-5'-tetraphosphate (dG4P-PEG). First, 2'-deoxyguanosine triphosphate (dGTP) was reacted with CDI in DMF to activate the terminal phosphate group, which was then reacted with dibutylammonium phosphate to give tetraphosphate. The terminal phosphate on this tetraphosphate was further activated with EDAC in 0.1M imidazole buffer, followed by reaction with diaminoheptane to provide the amino-linked tetraphosphate, which was further reacted with mPEG-NHS ester to provide the required four PEG-dG4P. After polymerase incorporation, the net charge on the released PEG is -3 (PEG-NH-triphosphate).

[0780] II. Testing modified nucleotides in single base extension reactions.

[0781] dG4P-PEG was characterized by MALDI-TOF mass spectrometry as shown in Table II.

[0782]

[0783] dG4P-PEG is an excellent substrate for...

example 3

[0785] Single-molecule detection of PEGs for labeling nucleotides through nanopores

[0786] Poly(ethylene glycol) is a non-electrolyte polymer that binds cations weakly (for example, it has a K of about 2M d Combine K + ion). Thus, the net charge on the polymer depends on the mobile cation concentration and on the presence of other moieties chemically linked to it. It has been demonstrated that a single α-hemolysin nanopore can readily discriminate between differently sized PEG polymers at a resolution better than monomer resolution (i.e., better than 44 g / mol) (Reiner et al. 2010; Robertson et al. 2007). That level of distinction is possible because polymers reduce the conductance of the pores due to volume exclusion (the pore conductance decreases with increasing polymer size) and by the incorporation of mobile cations (which would otherwise flow freely through the pores) (Reiner et al. 2010). In addition, the residence time of the polymer in the pores is very se...

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Abstract

This disclosure provides systems and methods for sequencing nucleic acids using nucleotide analogues and translocation of tags from incorporated nucleotide analogues through a nanopore. In aspects, this disclosure is related to composition, method, and system for sequencing a nucleic acid using tag molecules and detection of translocation through a nanopore of tags released from incorporation of the molecule.

Description

[0001] cross reference [0002] This application claims U.S. Provisional Application No. 61 / 781,353, filed March 14, 2013, U.S. Provisional Application No. 61 / 662,330, filed June 20, 2012, U.S. Provisional Application No. 61, filed June 20, 2012 / 662,329, U.S. Provisional Application No. 61 / 662,334, filed June 20, 2012, and U.S. Provisional Application No. 61 / 621,981, filed April 9, 2012, which are incorporated by reference in their entirety In this article. [0003] Statement Regarding Federally Sponsored Research [0004] This invention was made with government support under Grant No. HG005109 awarded by the National Institutes of Health. The government has certain rights in this invention. Background technique [0005] Nucleic acid sequencing is the process of determining the nucleic acid matrix of nucleic acids. The sequence information can aid in the diagnosis and / or treatment of the subject. For example, a subject's nucleic acid sequence can be used to identify, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/42
CPCB82Y10/00C12Q1/68C07H19/00C12Q1/48C12Q1/42G06F19/10G01N2333/9125G01N27/26C12Q1/6869C07H19/16C07H19/06C07H19/10C07H19/20C12Q2565/631B82Y5/00G01N33/48721C12Q2521/525C12Q2521/543C12Q2537/157C12Q1/6883C12Q2600/158C12Q2535/107
Inventor 静月·居希尔·库玛尔传娟·陶民辰·钱詹姆斯·J·拉索约翰·J·卡希恩沃维奇约瑟夫·W·F·罗伯森
Owner THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK
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