Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Reagents for reversibly terminating primer extension

A technology of components and bases, which is applied in the field of determining the components and processes of nucleic acid sequences, and can solve the problems of unanticipated practical tools and infeasibility of cyclic reversible termination sequencing methods.

Inactive Publication Date: 2013-03-13
动态组合化学技术有限责任公司
View PDF5 Cites 14 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] Unfortunately, US6664079 does not anticipate the utility of other aspects in sequencing using cycle reversible termination and is not feasible in the prior art

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Reagents for reversibly terminating primer extension
  • Reagents for reversibly terminating primer extension
  • Reagents for reversibly terminating primer extension

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0062] Example 1. TTP-ONH 2 Synthesis( figure 2 )

[0063] 3′-O-(N-acetone oxime)-thymine (1c).

[0064]3'-O-phthalimide-thymidine (1a) was prepared according to the following literature: [De Clercq, E., Inoue, I., Kondo, K. (1990) Preparation of 3-O- amino-2′-deoxyribonucleoside derivatives as antiviral agents for human retrovirus, particularly human immunodeficiency virus.Eur.Pat.Appl.14pp], [Kondo, K., Ogiku, T., Inoue, I. (1985) Synthesis of 5′ (3′)-O-amino nucleotides. Symp. Nucleic Acids Chem.16, 93-96], [Burgess, K., Gibbs, R.A., Metzker, M.L., Raghavachari, R. (1994) Synthesis of an oxyamide linked nucleotide dimer and incorporation into antisense oligonucleotide sequences.J.Chem.Soc.Chem.Commun.8,915-916], [Cook,P.D.,Sanghvi,Y.S.(1994)Preparation of antisense heteroatomic oligonucleotide analogs.PCT Int.Appl.90pp] .. Programs cited from these documents are expressly incorporated into this specification. This material (1.15 g, 3.0 mmol) was dissolved in an aqu...

example 2

[0072] The synthesis of example 2.dCTP-ONH2 ( image 3 )

[0073] 5'-O-Dimethoxytrityl-xylo-2'-deoxycytidine (2b).

[0074] in N 4 -Benzoyl-5′-O-Dimethoxytrityl-xylo-2′-deoxycytidine (2a, 8.9 g, 14 mmol), benzoic acid (2.5 g, 20 mmol) and triphenylphosphine (5.2 g, 20 mmol) in THF (150 mL), DIAD (3.7 mL, 20 mmol) was added at 0°C. The temperature of the reaction system was raised to room temperature overnight, and then, the reaction was quenched with water (0.5 mL). The reaction solvent was removed with a vacuum oil pump. Flash column chromatography (silica, eluent is ethyl acetate: n-hexane = 1: 1, finally 100% ethyl acetate) separation and purification, obtained N 4 -Benzoyl-3′-O-benzoyl-5′-O-dimethoxytrityl-xylo-2′-deoxycytidine (13.7 g) as a colorless foam containing substantial amounts of triphenylphosphine according to NMR oxides, and some elimination products (2',3'-alkenes). These intermediates were redissolved in methanol (450 mL) while treating with methanol...

example 3

[0088] Example 3. dATP-ONH 2 Synthesis( Figure 4 )

[0089] 5′-O-Dimethoxytrityl-xylo-2′-deoxyadenosine (3b) .

[0090]5′-O-dimethoxytrity-2′-deoxyadenosine (3a, 8.3 g, 15 mmol), benzoic acid (3.0 g, 24 mmol) and triphenylphosphine (6.5 g, 24 mmol) in THF (250 mL) solution, DIAD (4.5 mL, 24 mmol) was added at room temperature. After 1 hour, methanol (5 mL) was added to quench the reaction. The reaction solvent was removed by evacuation. Purification by flash column chromatography (silica, gradient 3% to 5% methanol in dichloromethane) afforded 3'-O-benzoyl-5'-O-dimethoxytrityl-xylo-2'-deoxy Adenosine (12 g) is a colorless foam. NMR showed that the product contained some triphenylphosphine oxide as well as some elimination products (2',3'-alkenes). These intermediates were dissolved in methanol (300 mL) and treated with sodium methoxide in methanol (5.3 mol, 4 mL, 21 mmol). After 16 hours, acetic acid (1.5 mL) was added to the reaction at room temperature. Vacuum wa...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

This invention relates to the field of nucleic acid chemistry, more specifically to the field of compositions of matter that comprise triphosphates of modified 2'-deoxynucleosides and oligonucleotides that are formed when these are appended to the 3'-end of a primer, wherein said modifications comprise NH2 moiety attached to their 3'-hydroxyl group and a fluorescent species in a form of a tag affixed to the nucleobase via a linker that can be cleaved. Such compositions and their associated processes enable and improve the sequencing of oligonucleotides using a strategy of cyclic reversible termination, as outlined in US Patent 6,664,079. Most specifically, the invention concerns compositions of matter that are 5'-triphosphates of ribo- and 2'- deoxyribonucleosides carrying detectable tags and oligonucleotides that might be derived from them. The invention also concerns processes wherein a DNA polymerase, RNA polymerase, or reverse transcriptase synthesizes said oligonucleotides via addition of said triphosphates to a primer.

Description

technical field [0001] The present invention relates to the field of nucleic acid chemistry, and more particularly to the components and processes used to determine the sequence of nucleic acids. Furthermore, the present invention relates to related components and processes thereof, which add a fluorescently labeled nucleotide to the 3'-end of a primer, and the 3'-hydroxyl of the resulting product oligonucleotide is temporarily blocked by a Group protection, the group can be deprotected in the subsequent process. Background technique [0002] "Sequencing by synthesis" as "sequencing using cycle reversible termination" (SuCRT) is a template-directed primer extension strategy. During the extension process, nucleoside triphosphates or thiotriphosphates are used as basic elements to add one Nucleotides. Polymerization is terminated after each nucleotide is added. At this time, primer extensions are checked to determine which type of nucleotide was added, which in turn deduces...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07H19/20C07H19/00C07H21/00
CPCC07H19/073C07H23/00C07H19/14C07H19/10C07H19/173
Inventor 史蒂芬·阿尔伯特·本纳丹尼尔·胡特尔妮科尔·欧罗拉·莱亚尔陈非
Owner 动态组合化学技术有限责任公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com