Hybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to-donor energy transfer system

• US 6,911,310 B2
• Filed: 11/29/2001
• Issued: 06/28/2005
• Est. Priority Date: 11/07/1991
• Status: Expired due to Fees

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: Heller, Michael J.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/997,374
• Publication Number:
  US 20030054361A1
• Time in Patent Office: 1,307 Days
• Field of Search: 425/6, 425/91.2, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: B01J 2219/00536 in the shape of disks
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: G06N 3/123 DNA computing
• CPC Class Codes: G11B 2007/24624 fluorescent dyes
• CPC Class Codes: G11B 7/244 comprising organic materials only
• CPC Class Codes: G11C 13/0014 comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 comprising bio-molecules
• CPC Class Codes: G11C 13/04 using optical elements ; using other beam accessed elements, e.g. electron or ion beam
• CPC Class Codes: H01L 24/95 at chip-level, i.e. with connecting carried out on a plurality of singulated devices, i.e. on diced chips
• CPC Class Codes: H01L 25/50 : Multistep manufacturing processes of assemblies consisting of devices, each device being of a type provided for in group H01L27/00 or H01L29/00 H01L21/50 takes precedence
• CPC Class Codes: H01L 2924/00 : Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
• CPC Class Codes: H01L 2924/01005 : Boron [B]
• CPC Class Codes: H01L 2924/01006 : Carbon [C]
• CPC Class Codes: H01L 2924/01011 : Sodium [Na]
• CPC Class Codes: H01L 2924/01012 : Magnesium [Mg]
• CPC Class Codes: H01L 2924/01013 : Aluminum [Al]
• CPC Class Codes: H01L 2924/01019 : Potassium [K]
• CPC Class Codes: H01L 2924/0102 : Calcium [Ca]
• CPC Class Codes: H01L 2924/01023 : Vanadium [V]
• CPC Class Codes: H01L 2924/01025 : Manganese [Mn]
• CPC Class Codes: H01L 2924/01032 : Germanium [Ge]
• CPC Class Codes: H01L 2924/01033 : Arsenic [As]
• CPC Class Codes: H01L 2924/01039 : Yttrium [Y]
• CPC Class Codes: H01L 2924/01043 : Technetium [Tc]
• CPC Class Codes: H01L 2924/01044 : Ruthenium [Ru]
• CPC Class Codes: H01L 2924/01054 : Xenon [Xe]
• CPC Class Codes: H01L 2924/0106 : Neodymium [Nd]
• CPC Class Codes: H01L 2924/01074 : Tungsten [W]
• CPC Class Codes: H01L 2924/01075 : Rhenium [Re]
• CPC Class Codes: H01L 2924/01076 : Osmium [Os]
• CPC Class Codes: H01L 2924/01077 : Iridium [Ir]
• CPC Class Codes: H01L 2924/01079 : Gold [Au]
• CPC Class Codes: H01L 2924/01082 : Lead [Pb]
• CPC Class Codes: H01L 2924/12042 : LASER
• CPC Class Codes: H01L 2924/12043 : Photo diode
• CPC Class Codes: H01L 2924/14 : Integrated circuits
• CPC Class Codes: Y10S 435/80 : Elimination or reduction of contamination by undersired ferments, e.g. aseptic cultivation
• CPC Class Codes: Y10S 436/80 : Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/13 : Tracers or tags

Abstract

The present invention contemplates monitoring the amplification of nucleic acid using chromophore-containing polynucleotides having at least two donor chromophores operatively linked to the polynucleotide by linker arms, such that the chromophores are positioned by linkage along the length of the polynucleotide at a donor-donor transfer distance, and at least one fluorescing acceptor chromophore operatively linked to the polynucleotide by a linker arm, such that the fluorescing acceptor chromophore is positioned by linkage at a donor-acceptor transfer distance from at least one of the donor chromophores, to form a photonic structure for collecting photonic energy and transferring the energy to an acceptor chromophore, and methods using the photonic structures.

Claims

1. A method for the amplification of a nucleic acid sequence, comprising the steps of:

providing a target nucleic acid sequence;

providing a first polynucleotide sequence having at least one donor chromophore, the first polynucleotide sequence being complementary to at least a portion of the target nucleic acid sequence;

providing a second polynucleotide sequence having at least one acceptor chromophore, the second polynucleotide sequence being complementary to a least a portion of the target sequence;

performing polymerase chain reaction to amplify the target nucleic acid sequence;

hybridizing the first and second polynucleotide sequences to the target nucleic acid sequence, such that when the first polynucleotide sequence and the second polynucleotide sequence are hybridized to the target nucleic acid sequence, the donor chromophore and acceptor chromophore are in an energy transfer relationship that substantially eliminates secondary quenching; and

irradiating the mixture to detect hybridizations of the first and second polynucleotide sequences by fluorescence energy transfer from the one or more donor chromophores of the first polynucleotide sequence to the one or more acceptor chromophores of the second polynucleotide sequence.

2. The method of claim 1, wherein the reaction mixture is a homogeneous format.

3. The method of claim 1, wherein the target nucleic acid comprises double stranded or denatured DNA.

4. The method of claim 1, wherein the mixture is irradiated for each PCR cycle.

5. The method of claim 1, wherein the target nucleic acid sequence comprises RNA.

6. The method of claim 1, wherein the target nucleic acid sequence comprises synthetic polynucleotide.

7. The method of claim 1, wherein the hybridization and amplification reactions occur entirely in solution.

8. The method of claim 1, wherein at least one of the first polynucleotide, the second polynucleotide, or the target nucleic acid is bound to a solid support or matrix that is insoluble in the analyte solution.

9. The method of claim 8, wherein the solid support or matrix is selected from the group consisting of glass, metals, silicon, organic polymers, membranes, and biopolymers.

10. The method of claim 1, wherein the polynucleotide sequence comprises a plurality of donor chromophores.

11. The method of claim 1, wherein the polynucleotide sequence comprises a plurality of acceptor chromophores.

12. The method of claim 1, further comprising the step of hybridizing another first polynucleotide sequence and another second polynucleotide sequence to the target nucleic acid sequence, wherein the donor chromophore and acceptor chromophore are in an energy transfer relationship.

13. The method of claim 12, further comprising the step of irradiating the mixture to detect hybridization of said another first and second polynucleotide sequences by fluorescence energy transfer from the one or more donor chromophore of said another first polynucleotide sequence to the one or more acceptor chromophore of said another second polynucleotide sequence.

14. The method of claim 1, wherein each of the donor chromophores and the acceptor chromophores is selected from the group consisting of 4,4′

-Diisothiocyanatodihydro-stilbene-2,2′

-disulfonic acid, 4-acetamido-4′

-isothiocyanato-stilbene-2,2′

-disulfonic acid, 4,4′

-Diisothiocyanatostilbene-2,2′

-disulfonic acid, Succinimidyl pyrene butyrate, Acridine isothiocyanate, 4-imethylaminophenylazophenyl-4′

-isothiocyanate (DABITC), Lucifer Yellow vinyl sulfone, Fluorescein isothiocyanate, Reactive Red 4 (Cibacron Brilliant Red 3B-A), Rhodamine X isothiocyanate, Texas Red (Sulforhodamine 101, sulfonyl chloride), Malachite Green isothiocyanate, or IR144.

15. The method of claim 1, wherein the step of providing a target nucleic acid sequence further comprises the steps of providing an mRNA sequence producing a double-stranded cDNA sequence from the mRNA sequence.

17. The method of claim 15, wherein the target nucleic acid comprises double stranded DNA.

18. The method of claim 15, wherein the target nucleic acid sequence comprises synthetic polynucleotide.

16. A method for the amplification of a nucleic acid sequence, comprising the steps of:

providing a target nucleic acid sequence;

providing a first polynucleotide sequence having at least one donor chromophore, the first polynucleotide sequence being complementary to at least a portion of the target nucleic acid sequence;

providing a second polynucleotide sequence having at least one acceptor chromophore, the second polynucleotide sequence being complementary to a least a portion of the target sequence;

providing four different nucleoside triphosphates, a thermostable amplification enzyme, and two primers, wherein the primers are substantially complementary to the target polynucleotide sequence;

denaturing the target nucleic acid sequence to form single stranded nucleic acids at an appropriate temperature;

hybridizing the first and second polynucleotide sequences to the target nucleic acid sequence, such that when the first polynucleotide sequence and the second polynucleotide sequence are hybridized to the target nucleic acid sequence, the donor chromophore and acceptor chromophore are in an energy transfer relationship that substantially eliminates secondary quenching;

irradiating the mixture to detect hybridizations of the first and second polynucleotide sequences by fluorescence energy transfer from the one or more donor chroniophores of the first polynucleotide sequence to the one or more acceptor chromophores of the second polynucleotide sequence; and

elongating the target polynucleotide sequence by adding the nucleotide triphosphates.

19. The method of claim 16, wherein the hybridization and amplification reactions occur entirely in solution.

20. The method of claim 16, wherein at least one of the first polynucleotide, the second polynucleotide, or the target nucleic acid is bound to a solid support or matrix that is insoluble in the analyte solution.

21. The method of claim 20, wherein the solid support or matrix is selected from the group consisting of glass, metals, silicon, organic polymers, membranes, and biopolymers.

22. The method of claim 16, wherein the polynucleotide sequence comprises a plurality of donor chromophores.

23. The method of claim 16, wherein the polynucleotide sequence comprises a plurality of acceptor chromophores.

24. The method of claim 16, wherein the step of providing a target nucleic acid sequence further comprises the steps of providing an mRNA sequence producing a double-stranded cDNA sequence from the mRNA sequence.