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Probe foranalysis of nucleic acids

A probe and nucleic acid technology, applied in the field of probes, can solve the problems of limited detection, reduced specificity of probe reaction, and unusable temperature.

Inactive Publication Date: 2008-07-02
LIGHTUP TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This approach presents two opposing design problems: high probe concentrations are required to obtain fast hybridization kinetics, but at the same time low probe concentrations are required to minimize background luminescence from free probes, which have been found to have neither Binds neither to TS nor to complementary probes
Other disadvantages are that probing is limited to a narrow temperature range because both the hybridization of the probe to TS and the secondary structure of the free probe must be stable
For example, temperatures at which TS does not hybridize to complementary NA cannot be used
Furthermore, the already significant thermal motion at room temperature reduces the quenching efficiency, so often even lower probing temperatures must be used, which in turn reduces the specificity of the probe response

Method used

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  • Probe foranalysis of nucleic acids
  • Probe foranalysis of nucleic acids
  • Probe foranalysis of nucleic acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 14

[0142] From Example 14 below:

[0143] • PNA-type probes have lower background signal than NA-type probes.

[0144] ·PNA-type probes get higher signal than NA-type probes when hybridized with TS.

[0145] • The signal response enhancement is significantly greater for PNA-type probes than for NA-type probes.

[0146] In addition, PNA probes have the following advantages over NA probes:

[0147] • PNA-type probes can be used at higher temperatures than NA-type probes because they form more stable complexes with TS. For example, the PP8-GCT-TO probe even showed a 35-fold signal increase at 45°C (Example 11), a temperature at which the corresponding NA-type probes do not form duplexes.

[0148] ·PNA-type probes can basically be used for any ionic strength, while the detection of NA-type probes is very sensitive to ionic strength. In fact, the comparison of Example 14 was performed under the condition of 500 mM NaCl in order to stabilize the NA-RG probe bound to TS.

[0149] •...

Embodiment 1

[0152] Example 1. Synthesis of RG.

[0153] dye

[0154] R 1 is the side chain containing the reactive group through which RG attaches to the SRE

Embodiment 2

[0155] Example 2. Synthesized probes.

[0156]

[0157]

[0158]

[0159] 1 (H) stands for free amino group and (NH 2 ) represents a terminal carboxamide.

[0160] 2 Rigid link base.

[0161] 3 Oligodeoxynucleotides with amino linkers were purchased from Scandinavian Gene Sythesis. The succinimidyl ester of TO was attached, and the probe was purified using the same procedure as the PNA-type probe in Example 4, except that TFA was replaced with 0.1 M TEAA (triethylammonium acetate), pH 7.0.

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Abstract

The invention is a probe for detecting nucleic acids having a particular sequence. It is composed of two joint units. One unit is chemically different from natural nucleic acids, but has the ability to recognize a particular sequence of bases or base pairs in single or double-stranded DNA of RNA. The other unit is a compound whose detectable properties are altered upon binding to nucleic acids.

Description

[0001] The present invention belongs to the class of probes that hybridize to nucleic acids. Such probes are useful in methods for the identification of specific genes, gene fragments, RNA molecules and other nucleic acids. These methods are mainly used clinically such as testing tissue, blood and urine samples, food engineering, agricultural and biological research. Background of the invention [0002] Probes that hybridize to nucleic acid (NA), by which we mean deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are used to determine the presence of specific target sequences (TS) in complex mixtures. Gillespie and Spiegelman (J. Mol. Biol. (Journal of Molecular Biology), 12, 829, 1956) first described the traditional hybridization method using a probe based on a reporter group (RG) (usually radioactive isotopes), and the method generally includes the following steps: immobilizing the nucleic acid to be tested on paper, glass beads or a plastic surface; adding excess pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C07H21/00C12Q1/68C07D417/06C12N15/09C07D417/14C07D473/00C07D487/04C09B23/02C12Q1/6818C12Q1/6825G01N33/58
CPCC07D417/14C07D417/06C07H21/00C07D487/04C12Q1/6818C12Q1/6825C09B23/02Y10T436/143333C12Q2565/1015C12Q2565/107
Inventor 米凯尔·库比斯塔尼克·斯范维克
Owner LIGHTUP TECH