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Detection of target nucleic acid by solid-phase molography

A target nucleic acid and nucleotide technology, applied in the field of polymerase chain reaction, can solve problems such as not easy multiplexing, improvement potential limit, etc.

Pending Publication Date: 2021-07-30
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As mentioned above, since different dyes are required for each target nucleic acid to be detected, these assay procedures cannot be easily multiplexed and thus their potential for improvement is limited

Method used

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  • Detection of target nucleic acid by solid-phase molography
  • Detection of target nucleic acid by solid-phase molography
  • Detection of target nucleic acid by solid-phase molography

Examples

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Effect test

example 1

[0053] Example 1 Feasibility Study: Cleavage and Extension of HIV-LTR Probes in Solution

[0054] Real-time PCR experiments are performed to verify that the tagged probe contains an annealing moiety that, in the presence of the target HIV-1 LTR gene, hybridizes to a portion of the HIV-1 LTR gene and that the unique tag moiety is cleaved; and The released tag portion will be able to anneal to and extend from a "moligo" template comprising a sequence complementary to the tag portion. Table 1 shows the nucleotide sequences of the tagged probes and moligos.

[0055] Table 1

[0056]

[0057] PCR assays were performed in individual wells in the presence (FL HIV(+)) or absence (FL HIV(-)) of the target HIV-1 LTR gene and in the presence or absence of the Moligo tag 3 template. At the end of the assay, aliquots were removed from the wells and subjected to HPLC chromatography to analyze the resulting product. The experimental results are shown in figure 2 in the chromatogram...

example 2

[0058] Example 2 Feasibility Study of Nucleic Acid Hybridization Detection by Molecular Imaging

[0059] A feasibility study for the detection of nucleic acid hybridization and extension by molecular imaging was performed on a Zeptochip microarray chip (Zeptosens AG, Witterswil, Switzerland) as described in Pawlak M. et al., Proteomics 2002;2(4):383-93 described, which is hereby incorporated by reference in its entirety. Will be made of 20mM Tris, 5mM MgCl 2 , 50 mM KCl, pH 8.0, was applied to the flow chamber inside the flow-through system of the chip at a constant flow rate of 15 μL / min. First, streptavidin (SAv) was injected onto the surface with a mass density modulation of 520 pg / mm as a normalized signal for focused molecular imaging 2 middle. A negative control between cDNA without an affinity tag and immobilized SAv did not produce any change in molecular imaging signal (see image 3 (i)), which indicates that no unwanted interactions have occurred. Next, a biot...

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Abstract

The present invention describes methods for performing real-time PCR for detection and quantitation of target nucleic acids using tagged oligonucleotide probes and solid-phase molography.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 782867, filed December 20, 2018, which is incorporated herein by reference in its entirety. [0003] Reference Sequence Listing [0004] This application contains a sequence listing filed as an electronic text file entitled "P34923-WO PCT fling Sequence Listing", 46kb in size and created on November 17, 2019. technical field [0005] The present invention relates to methods of polymerase chain reaction (PCR), and more particularly to methods for performing multiplex PCR and detecting target nucleic acids by solid-phase biosensors. Background technique [0006] Polymerase chain reaction (PCR) has become a ubiquitous tool in biomedical research, disease monitoring, and diagnosis. Amplification of nucleic acid sequences by PCR is described in US Patent Nos. 4,683,195, 4,683,202, and 4,965,188. PCR is now well known in the art and has...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825C12Q1/6853
CPCC12Q1/6825C12Q1/6853C12Q2521/319C12Q2525/161C12Q2531/113C12Q2565/519C12Q2565/607C12Q1/686
Inventor D·海因德尔N-P·斯蒂格勒A·灿I·科兹洛夫V·盖特丹
Owner F HOFFMANN LA ROCHE & CO AG
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