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

A polynucleotide and polychromophore technology, which is applied in the field of detecting and analyzing polynucleotides and compositions using multiple chromophores that acquire light, and can solve the problems of low yield, high cost, low detection sensitivity, etc.

Inactive Publication Date: 2012-10-03
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

7，8 Difficulty labeling two DNA loci results in low yields, high costs, and generates a single labeled impurity that makes detection less sensitive

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

[0089] Example 1. FRET identification of polychromophore / signaling chromophore pairs

[0090] Using cationic water-soluble conjugated polymer poly(9,9-bis(6'-N,N,N-trimethylammonium)-hexyl)-fluorene phenylene), i.e., prepared as described in 23 with iodide The polymer 1 of the counter anion, and the sensing peptide nucleic acid PNA-C* having the sequence 5'-CAGTCCAGTGATACG-3' and conjugated with fluorescein (C*) at the 5' position, verify the polychromatic emission of energy from harvesting light The ability of the chromophore system to transfer to the signaling chromophore on the sensing PNA. The respective absorption (green and orange) and emission (blue and red) spectra of polymer 1 and the sensing peptide nucleic acid PNA-C* are shown in figure 2 shown. 1 and PNA-C* achieve excitation at 380 and 480 nm, respectively. The data show that there is an optical window for the specific excitation of polymer 1. Furthermore, there is an excellent overlap between the emission o...

Embodiment 2

[0091] Example 2. Verification of FRET in the presence of target polynucleotides

[0092] Without polymer 1, in a separate container, make PNA-C* probe ([PNA-C*]=2.5×10 -8 M) contacted with an equimolar amount of complementary 15-base pair ssDNA, i.e. (5'-CGTATCACTGGACTG-3'), and made it in the same way with a non-complementary 15-base ssDNA, i.e. (5'-ACTGACGATAGACTG-3') )4 contacts. The annealing step is buffer-free, i.e. low ionic strength, and at a higher T than PNA-C* m 2°C lower conditions (72°C, 10 -8 M, pH=5.5). 32，33 A melting test was then performed and the absorbance was monitored at 260 nm using a UV / Vis spectrophotometer. 18 Elevated temperature can result in increased absorbance when the hybridized duplex melts in samples containing complementary ssDNA, since the absorbance of both single strands is much higher than that of the hybridized duplex. As expected, the sample containing non-complementary ssDNA did not show the aforementioned increase in absorbance ...

Embodiment 3

[0094] Example 3. Optimization of energy transfer

[0095] Energy transfer was optimized by varying the ratio of compound 1 to PNA-C*. at [PNA-C*]=2.5×10 -8 The initial addition of compound 1 can immediately increase the FRET ratio at the concentration of . A decrease in FRET was observed when [1] was much larger than [PNA-C*]. Based on previously published molecular weight data, the maximum value of the FRET ratio corresponds to an approximately 1:1 ratio of polymer chains to PNA chains. 23 This association is expected because when [1] / [PNA-C*]1 scheme, not all photons generated by 1 (donor) can be transferred to the DNA / PNA-C* hybrid (acceptor). It should be noted that the C* emission at the saturation point is more than 25 times higher than that obtained by direct C* excitation (480 nm), further demonstrating that the signal amplification is provided by the polychromophore structure of polymer 1.

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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 PNA complementary to the target polynucleotide. The sensor PNA comprises a signaling chromophore to absorb energy from the excited multichromophore and emit light 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] The work to obtain the present invention was supported by grants GM62958-01 from the National Department of Health, DMR-0097611 from the National Science Foundation, and N00014-98-1-0759 from the Office of Naval Research. The United States Government may have certain rights in this invention. technical field [0003] The present invention relates to methods, articles and compositions for the detection and analysis of polynucleotides in samples. Background of the Invention [0004] Methods that enable real-time detection of DNA sequences with high sensitivity are of significant academic and economic interest. 1，2，3 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, can be excited but lack sequence specificity when inserte...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C07H21/02G01N33/53C08G63/48C08G63/91G01NG01N21/78G01N33/543G01N33/566
CPCC12Q1/682C12Q1/6818Y10T436/143333C12Q2565/107C12Q2525/107C12Q2527/125B82B3/00B82Y5/00
Inventor G·C·巴赞B·S·盖洛德
Owner RGT UNIV OF CALIFORNIA
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