Chemical methods for producing tagged nucleotides

A nucleotide and tagged technology, which can be used in biochemical equipment and methods, organic chemistry, microbial measurement/testing, etc., and can solve the problem of insufficient signal difference

Active Publication Date: 2017-05-24
THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Likewise, pyrimidines (i.e., C, T, and U) have similar size, shape, and charge to each other, and in some cases provide signals that are not sufficiently different

Method used

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  • Chemical methods for producing tagged nucleotides
  • Chemical methods for producing tagged nucleotides
  • Chemical methods for producing tagged nucleotides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0268] Example 1 - Synthesis of Coumarin-PEG-dG4P Tagged Nucleotides

[0269] In this example, nucleotides were purified by reverse phase HPLC using a 150 x 4.6 mm column (Supelco), mobile phase: A, 8.6 mM Et 3 N / 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol in water (pH 8.1); B, methanol. Elution was performed as follows: 100% A isocratic for 10 minutes, followed by a linear gradient of 0-50% B for 20 minutes, then 50% B for 30 minutes.

[0270] Such as Figure 16 As shown in the scheme, Coumarin-PEG n The synthesis of -dG4P includes three synthetic operations: A, B and C.

[0271] A. 2'-Deoxyguanosine-5'-tetraphosphate (dG4P) and dG4P-NH 2 Synthesis: First, 2'-dG4P was synthesized from 2'-dGTP. 300 μmol of 2'-dGTP (triethylammonium salt) was converted to the tributylammonium salt by using a solution of 1.5 mmol (5 equivalents) of tributylamine in dry pyridine (5 ml). The resulting solution was concentrated to dryness and co-evaporated with 5 ml anhydrous DMF (x2). ...

Embodiment 2

[0281] Example 2 - Characterization of released tags by MALDI-TOF MS

[0282] After HPLC purification, the expected coumarin-PEG-NH was confirmed by MALDI-TOF-MS analysis 2 molecular( Figure 17 ). MALDI-TOF-MS results showed that the coumarin-PEG-NH produced by acid hydrolysis 2 The tag is identical to the released tag produced during the polymerase reaction following alkaline phosphatase treatment.

[0283] refer to Figure 17 , as shown by MALDI-TOF-MS analysis, by generating coumarin-PEG16-NH 2 Acid hydrolysis of coumarin-PEG16-dG4P yields coumarin-PEG20-NH 2 Acid hydrolysis of coumarin-PEG20-dG4P yields coumarin-PEG24-NH 2 Acid hydrolysis of coumarin-PEG24-dG4P and generation of coumarin-PEG36-NH 2 Coumarin-PEG-NH produced by acid hydrolysis of coumarin-PEG36-dG4P 2 Tag, identical to the corresponding release tag generated in the polymerase extension reaction after treatment with alkaline phosphatase. A composite image of four individually acquired MS spectra i...

Embodiment 3

[0284] Example 3—Detection of oligonucleotide labels

[0285] Nanopore array device (see for example Figure 12 ) for detecting 4 different current levels for 4 different labels. Such as Figure 18 As shown, each tag can be distinguished from any of the other three tags (ie, the histogram shows four distinct peaks labeled with the corresponding tag in the graph). Each tag is a "T" oligonucleotide homopolymer approximately 30 bases in length, biotinylated at the 3' end, with two regions in the potentially modified strand. In each 30-base long molecule, the modified regions were base positions 11, 12 and 13 and positions 17, 18 and 19 from the 3' end. As used herein, "x" is an abasic site (abasic), and "T" is thymine. The four tags are:

[0286] (a) "pseudo-label-XXX_XXX" has the sequence: streptavidin-biotin-10T-xxx-3T-xxx-11T (SEQID NO.1),

[0287] (b) "pseudo-tag-TTT_XXX" has the sequence: streptavidin-biotin-10T-TTT-3T-xxx-11T (SEQID NO.2),

[0288] (c) The "30T" ta...

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Abstract

This disclosure provides systems and methods for attaching nano pore-detectable tags to nucleotides. The disclosure also provides methods for sequencing nucleic acids using the disdosed tagged nudeotides. Provided herein are nudeotides with attached tags and methods for attaching tags to nudeotides. The tags can be attached by chemical reactions, such as 'dick chemistry'. In an aspect, the present disdosure provides a tagged nudeotide, comprising: (a) a poly-phosphate moiety having a terminal phosphate; and (b) a tag covalently coupled to the terminal phosphate of the nudeotide by a triazole, a 1,2-diazine, a disulfide, a secondary amine, a hydrazone, a thio-acetamide, or a maleimide-thioadduct.

Description

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61 / 969,628, filed March 24, 2014, the contents of which are incorporated herein by reference. [0002] This invention was made with Government support under Grant No. 5R01HG007415 awarded by the National Institutes of Health. The government has certain rights in this invention. [0003] This application incorporates by reference the nucleotide and / or amino acid sequences present in the file entitled "150323_0575_85625_Sequence Listing_JAK.txt", which is 52 kilobytes in size, filed March 23, 2015 as Generated in IBM-PC machine format, with compatibility with MS-Windows operating systems, included in the text file filed on March 23, 2015 as part of this application. technical field [0004] The present application relates to tagged nucleotide compositions, methods of making and using the disclosed tagged nucleotide compositions for sequencing nucleic acids, particularly na...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H19/04
CPCC07H19/10C07H19/20C07H17/02C12Q1/6869C12Q2565/631C12Q1/6806C12Q1/6874
Inventor 卡尔·W·富勒希夫·库马尔静岳·具兰德尔·戴维斯罗杰·陈
Owner THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK
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