Detection of nucleic acids

A target nucleic acid and detector technology, which is applied in the determination/inspection of microorganisms, measurement devices, preparation of samples for testing, etc., can solve problems such as inability to detect target nucleic acids

Inactive Publication Date: 2007-04-25
ORTHO-CLINICAL DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] The method described in the '984 patent can be used to detect the presence of double-stranded DNA, but cannot detect specific target nucleic acids

Method used

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  • Detection of nucleic acids
  • Detection of nucleic acids
  • Detection of nucleic acids

Examples

Experimental program
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Effect test

Embodiment 1

[0179] Detection of target DNA hybridized to particle-immobilized oligonucleotides: DNA-binding fluorophores

[0180]Hybridized target DNA is detected with a fluorophore that binds to dsDNA. The target DNA (SEQ ID NO: 2) in this example was hybridized to the bead-immobilized probe (SEQ ID NO: 1) in the presence of excess calf thymus DNA. Fluorophores are mixed with the hybridized target-bead suspension and analyzed by flow cytometry. The results using thiazole orange as the dsDNA binding fluorophore are shown in Figure 2A-F. Front azimuthal scatter (FW-SC) and right angle scatter (RT-SC) patterns of beads incubated with 1 μg of calf thymus DNA in the absence (Fig. 2A) and presence (Fig. 2B) of target DNA are shown. Light scatter gating of the beads allows analysis of particle sorting within a narrow range of FW-SC x RT-SC values ​​by means of image analysis software algorithms, thus eliminating particles outside that size range. Using thiazole orange as the dsDNA binding fl...

Embodiment 2

[0185] nuclease protection

[0186] Hybridization of the biotinylated particle-immobilized oligonucleotide probe (Oligo-1A) to the target oligonucleotide (SEQ ID: 2) protects the probe from hydrolysis by single strand specific DNA endonucleases. Particle-immobilized oligonucleotides and CTDNA were incubated together in the presence and absence of 1000 femole target DNA as described in Example 1, followed by incubation with S1 nuclease. Biotin is released upon endonuclease hydrolysis of the oligonucleotide probe unless it is protected from hydrolysis by hybridization to the target DNA.

[0187] Add 8 µl of an aliquot of streptavidin-linked fluorescer (streptavidin-phycoerythrin from Molecular Probes) diluted 1:10 in TE buffer to 16 µl of nuclease-treated Treated samples were incubated at room temperature for 10 min. The bound streptavidin-linked fluorescer serves as a reporter for the intact bead-linked oligonucleotide. The mixture was analyzed by flow cytometry. The mean c...

Embodiment 3

[0189] Quantification of target DNA: DNA-bound fluorophores

[0190] As shown in Figure 3, the fluorescence of the fluorophore sybr green bound to a hybrid of target DNA (SEQ ID NO: 2) and bead-immobilized oligonucleotide (SEQ ID NO: 1) varies with target copy number .

[0191] The uniqueness of the fluorescent signal associated with bead-immobilized oligonucleotides was dependent on the concentration, demonstrating that the target DNA content can be quantified over a concentration range of approximately 6 orders of magnitude. In the background of 0.8 μg of non-specific calf thymus DNA, it was clearly detected as low as the equivalent of 440 attomoles (attomoles) or about 2.5×10 8 25.7 picograms of target dsDNA were copied for the 90-mer target. Therefore the target DNA is at about 3.11 x 10 4 It is readily detectable in the presence of a fold excess of nonspecific DNA. The results also show that the method is very sensitive in the concentration range; about 1.25×10 8 to ...

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Abstract

Homogeneous and multiplex assays for target nucleic acids hybridized to oligonucleotide probes immobilized on particulate and non-particulate substrates are described. Target nucleic acids are determined using detection methods, such as flow cytometric methods, capable of particle discrimination based on the light scattering or fluorescence properties of the particle, or by spatial resolution of oligonucleotides immobilized at specific loci on a substrate. Target-correlated fluorescence signal, originating from a target nucleic acid hybridized to the substrate-immobilized oligonucleotide is determined as a measure of the presence or amount of the target nucleic acid.

Description

field of invention [0001] The present invention relates to the detection of nucleic acids. More precisely, the present invention relates to the use of support-immobilized oligonucleotides and providing detectable signals in association with said support or support-immobilized oligonucleotides and in association with target nucleic acids hybridized thereto. Signaling methods to detect target nucleic acids. Background of the invention [0002] Detection of nucleic acids is widely used to determine the presence and copy number of specific genes, known sequences, and to identify and quantify viruses, prokaryotic and eukaryotic pathogens in clinical and environmental samples. An important characteristic of nucleic acids is their ability to form sequence-specific hydrogen bonds with nucleic acids having complementary nucleotide sequences. This ability of nucleic acids to hybridize to their complementary strands has been used to advantage in techniques known as hybridization assa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C07H21/04G01N31/22C12M1/36C12N15/09C12Q1/6816C12Q1/6837C12Q1/686G01N1/30G01N33/542G01N33/543
CPCG01N33/54346C12Q1/6816C12Q1/6837G01N33/542C12Q1/686G01N1/30C12Q2563/173C12Q2563/149C12Q2565/626C12Q1/68
Inventor M·C·康内利G·塞里诺T·J·梅尔科利诺
Owner ORTHO-CLINICAL DIAGNOSTICS
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