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Methods and compositions for detection and analysis of polynucleotides using light harvesting multichromophores

A polynucleotide and multiple chromophore technology, applied in the field of detection and analysis of polynucleotides, can solve problems such as high cost, low yield, and reduced detection sensitivity

Inactive Publication Date: 2010-12-15
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Difficulty in labeling both DNA loci leads to low yields, high cost and impurities of single label, which reduces detection sensitivity 9

Method used

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  • Methods and compositions for detection and analysis of polynucleotides using light harvesting multichromophores
  • Methods and compositions for detection and analysis of polynucleotides using light harvesting multichromophores
  • Methods and compositions for detection and analysis of polynucleotides using light harvesting multichromophores

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1. Identification of FRET protocols

[0095] Demonstration using a cationic water-soluble conjugated polymer poly(9,9-bis(6′-N,N,N-trimethylammonium)-hexyl)-phenylenefluorene), a polymer 1 with an iodide counter anion Hybridization assays utilizing energy transfer from a light-harvesting multichromophore system to a signaling chromophore. The sensor polynucleotide sequence is 5'-GTAAATGGTGTTAGGGTTGC-3', corresponding to anthrax (Bacillus anthracis (Bacillus anthracis)) spore-coated plasmid pX02, with fluorescein at the 5' position to form oligo-C ＊ an example of 24 . The absorption and emission spectra of the polymer and the signal chromophore are shown in figure 1 middle. The data show an optical window for polymer 1 excitation between the absorption of DNA and fluorescein. Such as figure 1 As shown, direct excitation of polymer 1 results in energy transfer (ET) to fluorescein. The absorption overlap of fluorescein and polymer 1 excitation is selected to...

Embodiment 2

[0096] Example 2. Demonstration of FRET in the presence of target polynucleotides

[0097] Oligo-C ＊ Hybridization of the probes results in a change in the ET ratio. In equimolar amounts of a 40 base pair strand containing the complementary 20 base pair sequence 5'-CATCTGTAAATCCAAGGTAGCAACCCTAACACCATTTAC-3', and the same pattern of non-complementary 40 with the sequence 5'-AAAATATTGTGTATCAAAATGTAAATGGTGTTAGGGTTGC-3' In the presence of a base chain, the sensor polynucleotide ([oligo-C ＊ ]=2.1×10 -8 M) at lower than its T m (58.4°C) Annealed at 2°C. A direct comparison of the resulting fluorescence revealed a 6-fold greater ET ratio than hybridized DNA. see figure 2 . It is also highly significant that these optical differences were observed in the presence of 10 mmol sodium citrate and 100 mmol sodium chloride buffer. The buffer ion masks the charge on the complementary DNA, which promotes hybridization but weakens the electrostatic interaction between CPs and the oppo...

Embodiment 3

[0098] Example 3. Optimization of energy transfer

[0099] By changing polymer 1 with oligo-C ＊ ratio to optimize energy transfer. in [oligo-C ＊ ]=2.1×10 -8 At a concentration of M, the initial addition of polymer causes an immediate increase in the ET ratio, when the amount of polymer 1 begins to far exceed the oligo-C ＊ The ET ratio decreases when the amount is increased. The maximum value of the ET ratio corresponds to the polymer chain to oligo-C ＊ almost a 1:1 ratio. Not every chain is tightly complexed to oligo-C at high polymer concentrations ＊ and the donor emission rises faster than the signal chromophore emission. Such a relationship is expected because when [polymer 1] / [oligo-C ＊ ] ＊ ]>1, not all photons bound by polymer 1 (donor) can be transferred to the oligo-C ＊ (receptor). Once polymer-oligo-C ＊As the repeat unit reaches approximately 100, the photoluminescence of the reporter chromophore is no longer enhanced, indicating saturation of the acceptor. ...

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Abstract

Methods, compositions and articles of manufacture for assaying a sample for a target polynucleotide are provided. A sample suspected of containing the target polynucleotide is contacted with a polycationic multichromophore and a sensor polynucleotide complementary to the target polynucleotide. The sensor polynucleotide comprises a signaling chromophore to receive energy from the excited multichromophore and increase emission in the presence of the target polynucleotide. The methods can be used in multiplex form. Kits comprising reagents for performing such methods are also provided.

Description

[0001] Statement Regarding Federally Funded Research [0002] Work leading to the present invention was performed under Grant No. GM6295801 from the National Institutes of Health, Grant No. DMR-0097611 from the National Science Foundation, and Grant No. N00014-98-1-0759 from the Office of Naval Research. The US Government may have limited rights in this invention. technical field [0003] The present invention relates to methods, goods and compositions for the detection and analysis of polynucleotides in samples. Background of the invention [0004] Methods allowing real-time and high-sensitivity detection of DNA sequences are of great scientific and economic interest 1，2，3 . Their applications include medical diagnostics, identification of genetic mutations, gene delivery monitoring, and specific genomic technologies 4 . Cationic organic dyes, such as ethidium bromide and thiazole orange, emit when inserted into the groove of double-stranded DNA (dsDNA) and function as ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C07H21/00C07H21/02C07H21/04C12NG01N21/00G01N21/64
CPCC12Q1/6818C12Q1/6813Y10T436/143333C12Q2565/107C12Q2563/107C12Q2523/313C12Q1/6825C12Q1/6834
Inventor G·C·巴赞B·S·盖洛德S·王
Owner RGT UNIV OF CALIFORNIA