EP1322785A4 - Kombinatorische fluoreszenzenergietransfer-tags und deren verwendungen - Google Patents

Kombinatorische fluoreszenzenergietransfer-tags und deren verwendungen

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Publication number
EP1322785A4
EP1322785A4 EP01968905A EP01968905A EP1322785A4 EP 1322785 A4 EP1322785 A4 EP 1322785A4 EP 01968905 A EP01968905 A EP 01968905A EP 01968905 A EP01968905 A EP 01968905A EP 1322785 A4 EP1322785 A4 EP 1322785A4
Authority
EP
European Patent Office
Prior art keywords
composition
nucleic acid
oligonucleotide
matter
fluorophore
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01968905A
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English (en)
French (fr)
Other versions
EP1322785A1 (de
Inventor
Jingyue Ju
Zengmin Li
Anthony Tong
James J Russo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Columbia University in the City of New York
Original Assignee
Columbia University in the City of New York
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Filing date
Publication date
Priority claimed from US09/658,077 external-priority patent/US6627748B1/en
Application filed by Columbia University in the City of New York filed Critical Columbia University in the City of New York
Publication of EP1322785A1 publication Critical patent/EP1322785A1/de
Publication of EP1322785A4 publication Critical patent/EP1322785A4/de
Withdrawn legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y10/00—Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures

Definitions

  • M-FISH multi-color fluorescence in situ hybridization
  • This invention provides a composition of matter comprising multiple fluorophores, each of which is bound to a molecular scaffold at a separate predetermined position on the scaffold, such separate predetermined positions being selected so as to permit fluorescence energy transfer between one such fluorophore and another such fluorophore, wherein the one such fluorophore and the another such fluorophore are characterized by the maximum emission wavelength of one being greater than the minimum excitation wavelength of the other.
  • This invention further provides the instant composition of matter comprising two fluorophores, each of which is bound to a molecular scaffold, at a separate predetermined position on the scaffold, such separate positions being selected so as to permit fluorescence energy transfer between such fluorophores, and such fluorophores being characterized by the maximum emission wavelength of one of the fluorophores being greater than the minimum excitation wavelength of the other fluorophore .
  • This invention further provides the instant composition of matter comprising three fluorophores each of which is bound to a molecular scaffold at- a separate predetermined position on the scaffold, such separate predetermined positions being selected so as to permit fluorescence energy transfer among such fluorophores and such fluorophores being characterized by the maximum emission wavelength of one such fluorophore being greater than the minimum excitation wavelength of the second such fluorophore and the maximum emission wavelength of such second fluorophore being greater than the minimum excitation wavelength of the third such fluorophore.
  • This invention further provides the instant composition of matter, wherein each fluorophore is covalently bound to the molecular scaffold.
  • This invention further provides the instant composition of matter, wherein the efficiency of the fluorescence energy transfer is less than 20%.
  • This invention further provides the instant composition of matter, wherein the molecular scaffold is rigid.
  • This invention further provides the instant composition of matter, wherein the molecular scaffold is polymeric.
  • This invention further provides the instant composition of matter, wherein the molecular scaffold comprises a nucleic acid.
  • This invention further provides the instant composition of matter, wherein the molecular scaffold comprises a peptide.
  • This invention further provides the instant composition of matter, wherein the molecular scaffold comprises a polyphosphate .
  • This invention further provides the instant composition of matter, wherein at least one fluorophore is a fluorescent dye.
  • This invention further provides the instant composition of matter, wherein the fluorescent dye is 6-carboxyfluorescein .
  • This invention further provides the instant composition of matter, wherein the fluorescent dye is N , N , N ' , N ' -tetramethyl-6-carboxyrhodamine .
  • This invention further provides the instant composition of matter, wherein the fluorescent dye is cyanine-5 monofunctional dye.
  • This invention further provides the instant composition of matter, wherein at least one fluorophore is a luminescent molecule.
  • This invention further provides the instant composition of matter, wherein at least one fluorophore is a quantum dot.
  • This invention also provides a composition of matter having the structure:
  • S represents a 1 ' , 2 ' -dideoxy ⁇ bose phosphate moiety
  • m is an integer greater than 1 and less than 100
  • each T represents a thymidine derivative
  • FAM represents 6-carboxyfluorescein derivative
  • TAM represents N, , N' , ' -tetramethyl- 6-carboxyrhodam ⁇ ne derivative
  • each solid line represents a covalent bond
  • R represents either a hydroxy or phosphate terminus
  • Q represents either a hydroxy or phosphate terminus, with the proviso that R and Q are different.
  • This invention further provides the instant composition of matter, wherein m is .
  • This invention further provides the instant composition of matter, wherein m is 6.
  • This invention further provides the instant composition of matter, wherein m is 9. This invention further provides the instant composition of matter, wherein m is 13.
  • This invention also provides a composition of matter having the structure:
  • S represents a 1 ' , 2 ' -dideoxyribose phosphate moiety
  • m is an integer greater than 1 and less than 100
  • T represents a thymidine derivative
  • FAM represents a 6-carboxyfluorescein derivative
  • Cy5 represents a cyanine-5 monofunctional dye derivative
  • each solid line represents a covalent bond
  • R represents either a hydroxy or phosphate terminus
  • Q represents either a hydroxy or phosphate terminus, with the proviso that R and Q are different.
  • This invention further provides the instant composition of matter, wherein m is 4.
  • This invention further provides the instant composition of matter, wherein m is 5. This invention further provides the instant composition of matter, wherein m is 7.
  • This invention further provides the instant composition of matter, wherein m is 10.
  • This invention further provides the instant composition of matter, wherein m is 13.
  • This invention also provides a composition of matter comprising the structure shown below:
  • S represents a 1 ' , 2 ' -dideoxyribose phosphate moiety
  • m is an integer greater than 1 and less than 100
  • n is an integer greater than 1' and less than 100
  • T represents a thy idine derivative
  • FAM represents a 6-carboxyfluorescein derivative
  • Cy5 represents a cyanine-5 monofunctional dye derivative
  • TAM represents a N,N, N' , N' -tetramethyl-6-carboxyrhodamine derivative
  • each solid line represents a covalent bond
  • R represents either a hydroxy or phosphate terminus
  • Q represents either a hydroxy or phosphate terminus, with the proviso that R and Q are different.
  • This invention further provides the instant composition of matter, wherein m is 3, and n is .
  • This invention further provides the instant composition of matter, wherein m is 4, and n is 6.
  • This invention further provides the instant composition of matter, wherein m is 5, and n is 5
  • This invention further provides the instant composition of matter, wherein m is 6, and n is 6.
  • This invention further provides the instant composition of matter, wherein m is 7, and n is 7.
  • This invention also provides a composition of matter comprising the structure shown below:
  • S represents a 1 ' , 2 ' -dideoxyribose phosphate moiety
  • m represents an integer greater than 1 and less than 100
  • T represents a thymidine derivative
  • TAM represents a N, N, N' , N' -tetramethyl-6-carboxyrhodamine derivative
  • each solid line represents a covalent bond
  • R represents either a hydroxy or phosphate terminus
  • Q represents either a hydroxy or phosphate terminus, with the proviso that R and Q are different.
  • This invention further provides the instant composition of matter, wherein m is .
  • This invention also provides a nucleic acid labeled with any of the instant compositions .
  • This invention provides any of the instant compositions, wherein the nucleic acid is DNA.
  • This invention provides any of the instant compositions, wherein the nucleic acid is RNA.
  • This invention provides any of the instant compositions, wherein the nucleic acid is DNA/RNA.
  • This invention also provides a method of determining whether a preselected nucleotide residue is present at a predetermined position within a nucleic acid comprising the steps of:
  • This invention further provides a method of determining whether at various predetermined positions within a nucleic acid, a preselected nucleotide residue is present at such position, wherein the preselected nucleotide residue may vary at different predetermined positions which comprises determining whether each preselected nucleotide is present each predetermined position according to the instant method.
  • This invention provides the instant method, wherein the presence of a plurality of given nucleotide residues is determined simultaneously.
  • This invention further provides the instant method, wherein the DNA ligase is Taq DNA ligase.
  • This invention further provides the instant method, wherein the second oligonucleotide has an isolation- permitting moiety affixed thereto, and wherein the method further comprises the steps of isolating the moiety-containing molecules resulting from step (a) and determining the presence therein of ligated first and second oligonucleotides.
  • This invention further provides the instant method, wherein the composition of matter affixed to the first oligonucleotide has a predetermined emission spectrum, and wherein the observation of this emission spectrum is employed to determine the presence of ligated first and second oligonucleotides in step (b) .
  • This invention also provides a method of determining whether a. preselected nucleotide residue is present at a predetermined position within a nucleic acid comprising the steps of:
  • This invention further provides a method of determining whether at various predetermined positions within a nucleic acid, a preselected nucleotide residue is present at such position, wherein the preselected nucleotide residue may vary at different predetermined positions which comprises determining whether each preselected nucleotide is present each predetermined position according to the instant method.
  • This invention further provides the instant method, wherein the DNA polymerase is thermo sequenase.
  • This invention further provides the instant method, wherein the dideoxynucleotide is selected from the group consisting of dideoxyadenosine triphosphate, dideoxycytidine triphosphate, dideoxyguanosine triphosphate, dideoxythymidine triphosphate, and dideoxyuridine triphosphate .
  • This invention further provides the instant method, wherein the composition of matter affixed to the oligonucleotide has a predetermined emission spectrum, and wherein the observation of this emission spectrum is employed to determine the presence of polymerization product in step (b) .
  • This invention further provides the instant methods, wherein observing the predetermined emission spectrum is performed using radiation having a wavelength of between 200 and lOOOnm.
  • This invention further ⁇ provides the instant methods, wherein the radiation has a wavelength of 488 n .
  • This invention further provides the instant methods, wherein observing the predetermined emission spectrum is performed using radiation having a bandwidth of between 1 and 50nm.
  • This invention further provides the instant methods, wherein the radiation bandwidth is lnm.
  • This invention further provides the instant methods, wherein the isolation-permitting moiety comprises biotin, streptavidin, phenylboronic acid, salicylhydroxa ic acid, an antibody or an antigen.
  • This invention further provides the instant methods, wherein the isolation-permitting moiety is attached to the oligonucleotide via a linker molecule.
  • This invention further provides the instant methods, wherein the isolation-permitting moiety is attached to the dideoxynucleotide via a linker molecule.
  • This invention further provides the instant methods, wherein the linker molecule is chemically cleavable.
  • This invention further provides the instant methods, wherein the linker molecule is photocleavable .
  • This invention further provides the instant methods, wherein the linker molecule has the structure:
  • FIG. 1A-B (A) Schematic of a multi-chromophore assembly connected to a linker.
  • 1 to n chromophores can be attached to the assembly with the chromophores separated by spacers as shown.
  • Chromophores can be, but not limited to, fluorescent dyes, quantum dots or luminescent molecules such as terbium chelate.
  • spacers such as nucleotides, peptides, a polymer linker formed by 1', 2 ' -dideoxyribose phosphates or other chemical moieties can be used.
  • the assembly label shown here is connected to a linker which can be designed as nucleic acids, proteins or cells, etc for multiplex biological assays.
  • F-4-T-6-C The numbers in F-4-T-6-C refer to the number of spacing nucleotides in the scaffold between dyes F and T, and T and C.
  • Figure 2A-D Spectroscopic data for tags F-4-T-6-C and F-7-T-3-C.
  • FIG. 3A-B Schematic labeling approach to construct CFET-primers and CFET-dUTPs .
  • the spacer between dyes is 1' , 2' -dideoxyribose phosphate (S) in (A) and proline (P) in (B) .
  • S 1' , 2' -dideoxyribose phosphate
  • P proline
  • m and "n” refer to the number of molecules in the spacer.
  • dUTP deoxyuridine triphosphate.
  • FIG. 4 The synthesis of CFET-dUTP.
  • the CFET tag comprises three different fluorescent dyes: Fam, Tam and Cy5.
  • Figure 5 Structures of Aminoallyl (AA) -dUTP, Fam- proline, and N-Hydroxy succinimide (NHS) esters of TAM and Cy5.
  • AA Aminoallyl
  • Fam- proline Fam- proline
  • NHS N-Hydroxy succinimide
  • Figure 6 Synthetic schemes to prepare Fam-proline, Azido-proline and Cy5-phosphine .
  • TMSCI trimethylsilyl chloride.
  • Figure 7 The eight unique fluorescence signatures of CFET tags generated in a three-color CAE system. FAM channel (520 ⁇ 20 nm, dotted line), TAM channel (585
  • Primer pairs are generated surrounding a base that can be mutated.
  • the wild-type primer is labeled with one CFET tag (Tag 1) and the mutation-specific primer with another CFET tag (Tag 2) .
  • Figure 9 Schematic of expected results from screening four potential mutation sites of Rbl gene using eight unique CFET Tags and the ligase chain reaction assay. Only ligation products are shown on the gels.
  • Figure 10 Schematic of chromosomal studies to detect macrodeletions and amplifications.
  • FIG. 11 This figure schematically shows the procedure for multiplex SNP detection through the ligation of hybridized CFET-labeled and biotinylated oligonucleotides .
  • Taq DNA ligase seals the nick between the two hybridized oligonucleotides if the nucleotides at the ligating junction are correctly base-paired to the template (A to T; C to G) .
  • CFET- labeled, biotinylated ligation products are then isolated using streptavidin-coated magnetic beads . After washing and releasing from the magnetic beads, the ligation products are electrokinetically injected into a three-color CAE system.
  • Each CFET-labeled ligation product which identifies a unique SNP, is unambiguously detected due to its distinct mobility and fluorescence signature in the CAE electropherogram.
  • Figure 12A-B Electropherogram of CFET-labeled ligation products for SNPs identification on exon 20 of the RBI.
  • A Detection of six nucleotide variations from synthetic DNA templates.
  • FAM ( T) and F-10-Cy5 ( T) peaks are obtained from two different locations of the same template.
  • F-9-T (C) and F-13-T ( T) peaks indicate mutations from the same locus of a DNA template, while F-4-T-6-Cy5 (A) and F-7-T-7-Cy5
  • (C) peaks identify mutations from the same locus of another DNA template.
  • (B) Detection of three homozygous genotypes (T, C and A) from a PCR product of RBI.
  • FIG. 13 This figure is a schematic of single base primer extension for multiplex SNP detection by using dye-labeled primers and biotinylated dideoxynucleoside triphosphates (ddNTP-Biotin) .
  • DNA template containing polymorphic sites is incubated with a dye-labeled primer, hybridizing the template adjacent to the polymorphic site, ddNTP-Biotin and thermo sequenase.
  • the primer extension products are analyzed for fluorescence signatures.
  • Figure 14 Three unique fluorescence signatures generated from dye-labeled extension products. FAM channel (light) and TAM channel (Dark) . The fluorescence signatures in the electropherograms were obtained by excitation at 488nm and the single base extension of the dye-labeled primers. The digital ratio denoting the fluorescence signature for each from the two detection channels is shown in parentheses .
  • Figure 15A-C The electropherograms of CFET-labeled primer extension products for multiplex SNPs identification on the mimic of exon 20 of the RBI .
  • FAM channel Light line
  • TAM channel Dark line
  • A) Detection of two individual homozygous genotypes from a wild type template.
  • FAM (T) and F-9-T (C) peaks were obtained from two different loci on the template.
  • B) Similar to (A) except a mutated template was used.
  • C Simultaneous detection of three nucleotide variations.
  • FAM (T) peak was obtained from a locus of the template where a homozygous genotype was found.
  • F-9-T (C) and F-13-T (T) peaks indicate the mutation R661W (heterozygote) from the same locus of a DNA template.
  • Figure 16 Schematic of a high throughput channel based, moiety-based purification system.
  • Sample solutions can be pushed back and forth between the two plates through glass capillaries and the coated channels in the chip, the channels being coated with an appropriate chemical to bind the moiety tag on the samples, e.g. streptavidin coating in the case of biotinylated oligonucleotides.
  • an appropriate chemical to bind the moiety tag on the samples e.g. streptavidin coating in the case of biotinylated oligonucleotides.
  • the moieties are attached by cleavable linkers, e.g. photocleavable linkers, the whole chip can be irradiated to cleave the samples after immobilization.
  • RBI Retinoblastoma gene
  • SNP Single nucleotide polymorphism
  • “Chemically cleavable” shall mean cleavable by any chemical means including but not limited to pH and temperature.
  • DNA/RNA shall mean a nucleic acid molecule comprising both deoxyribonucleotides and ribonucleotides .
  • Emission spectrum shall mean the amplitude and frequency of energy emitted from a composition of matter as a result of exciting radiation thereon.
  • Fluxible when used to describe a molecular scaffold, shall mean that the distance between the centers of any pair of fluorophores covalently bound to the scaffold varies by more than 50%.
  • Fluorescence energy transfer shall mean the transfer of energy between two fluorophores via a dipole-dipole interaction.
  • Fluorescent dye shall mean an organic dye molecule capable of emitting fluorescent energy of wavelength between 200 and lOOOnm when excited by an energy of shorter wavelength wherein the emitted energy results from a singlet to singlet transition. Examples are 6- carboxyfluorescein, N, N, N' , N' -tetramethyl-6- carboxyrhodamine, and cyanine-5 monofunctional dye.
  • Fluorophore shall mean a molecule, such as a fluorescent dye, quantum dot or luminescent molecule, capable of emitting energy of wavelength between 400 and lOOOnm when excited by an energy of shorter wavelength than the corresponding emission wavelength.
  • fluorophores include 6- carboxyfluorescein, N, N, N ' , N' -tetramethyl-6-carboxy rhodamine, cyanine-5 monofunctional dye, zinc sulfide-capped cadmium selenide quantum dots, and lanthanide chelates .
  • Hybridize shall mean the annealing of one single- stranded nucleic acid molecule to another single stranded nucleic acid molecule based on sequence complementarity.
  • the propensity for hybridization between nucleic acids depends on the temperature and ionic strength of their milieu, the length of the nucleic acids and the degree of complementarity. The effect of these parameters on hybridization is well known in the art (see Sambrook, 1989) .
  • Isolation-permitting moieties shall include without limitation biotin or streptavidin which bind to one another, antibodies or antigens which bind to one another, phenylboronic acid or salicylhydroxamic acid which bind to one another.
  • “Ligation-permitting conditions” include without limitation conditions of temperature, ionic strength, ionic composition, molecular composition, orientation and viscosity that allow one oligonucleotide to be joined enzymatically to another via a phosphodiester bond.
  • “Ligation” shall mean the enzymatic covalent joining of a nucleic acid with either another nucleic acid or a single nucleotide.
  • Linker molecule shall mean a chemical group used to covalently join two other molecules.
  • An example of a linker molecule is the structure given below:
  • Luminescent molecule shall mean a molecule capable of emitting energy of wavelength between 200 and lOOOnm when excited by energy of shorter wavelength than the corresponding emission wavelength, wherein the emitted energy does not result from a singlet to singlet transition.
  • luminescent molecules include europium polycarboxylate chelate and terbium chelates .
  • Molecular scaffold shall mean a molecular structure to which two or more fluorophores can be, and/or are, covalently bound at discrete loci thereon.
  • a molecular scaffold is polymeric, comprising monomeric units to which fluorophores can be bound.
  • the monomeric units which make up such polymeric scaffold can, but need not be, identical. Examples of such monomeric units include 1' , 2' -dideoxyribose phosphate and thymidine .
  • Nucleic acid molecule shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof.
  • the nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, New Jersey, USA) .
  • Oligonucleotide shall mean a nucleic acid comprising two or more nucleotides.
  • Photocleavable shall mean cleavable by electromagnetic energy of between 200 and lOOOnm wavelength .
  • Polymeric shall describe a molecule composed of more than two monomeric units.
  • Quantum dot shall mean a nanometer-sized composition of matter comprising a semi-conductor or metal, wherein such composition is capable of luminescence.
  • quantum dots include zinc- sulfide-capped cadmium selenide quantum dots.
  • Rigid when used to describe a molecular scaffold, shall mean that the distance between the centers of any pair of fluorophores covalently bound to the scaffold does not vary more than 50%.
  • This invention provides a composition of matter comprising multiple fluorophores, each of which is bound to a molecular scaffold at a separate predetermined position on the scaffold, such separate predetermined positions being selected so as to permit fluorescence energy transfer between one such fluorophore and another such fluorophore, wherein the one such fluorophore and the another such fluorophore are characterized by the maximum emission wavelength of one being greater than the minimum excitation wavelength of the other.
  • This invention further provides the instant composition of matter comprising two fluorophores, each of which is bound to a molecular scaffold, at a separate predetermined position on the scaffold, such separate positions being selected so as to permit fluorescence energy transfer between such fluorophores, and such fluorophores being characterized by the maximum emission wavelength of one of the fluorophores being greater than the minimum excitation wavelength of the other fluorophore.
  • This invention further provides the instant composition of matter comprising three fluorophores each of which is bound to a molecular scaffold at a separate predetermined position on the scaffold, such separate predetermined positions being selected so as to permit fluorescence energy transfer among such fluorophores and such fluorophores being characterized by the maximum emission wavelength of one such fluorophore being greater than the minimum excitation wavelength of the second such fluorophore and the maximum emission wavelength of such second fluorophore being greater than the minimum excitation wavelength of the third such fluorophore.
  • each fluorophore is covalently bound to the molecular scaffold.
  • the efficiency of the fluorescence energy transfer is less than 20%. In one embodiment the molecular scaffold is rigid.
  • the molecular scaffold is polymeric .
  • the molecular scaffold comprises a nucleic acid.
  • the molecular scaffold comprises a peptide.
  • the molecular scaffold comprises a polyphosphate .
  • At least one fluorophore is a fluorescent dye.
  • the fluorescent dye is 6- car oxyfluorescein.
  • the fluorescent dye is N,N,N',N'- tetramethyl-6-carboxyrhodamine .
  • the fluorescent dye is cyanine-5 monofunctional dye.
  • At least one fluorophore is a luminescent molecule.
  • At least one fluorophore is a quantum dot .
  • This invention also provides a composition of matter having the structure:
  • S represents a 1 ' , 2 ' -dideoxyribose phosphate moiety
  • m is an integer greater than 1 and less than 100
  • each T represents a thymidine derivative
  • FAM represents 6-carboxyfluorescein derivative
  • TAM represents N, , N' ,N' -tetramethyl- 6-carboxyrhodamine derivative
  • each solid line represents a covalent bond
  • R represents either a hydroxy or phosphate terminus
  • Q represents either a hydroxy or phosphate terminus, with the proviso that R and Q are different.
  • m is 4. In one embodiment m is 6. In one embodiment m is 9. In one embodiment m is 13.
  • This invention also provides a composition of matter having the structure:
  • S represents a 1 ', 2 ' -dideoxyribose phosphate moiety
  • m is an integer greater than 1 and less than 100
  • T represents a thymidine derivative
  • FAM represents a 6-carboxyfluorescein derivative
  • Cy5 represents a cyanine-5 monofunctional dye derivative
  • each solid line represents a covalent bond
  • R represents either a hydroxy or phosphate terminus
  • Q represents either a hydroxy or phosphate terminus, with the proviso that R and Q are different.
  • m is 4. In one embodiment m is 5. In one embodiment n m is 7. In one embodiment m is 10. In one embodiment m is 13.
  • This invention also provides a composition of matter comprising the structure shown below:
  • S represents a 1 ', 2 ' -dideoxyribose phosphate moiety
  • m is an integer greater than 1 and less than 100
  • n is an integer greater than 1 and less than 100
  • T represents a thymidine derivative
  • FAM represents a 6-carboxyfluorescein derivative
  • Cy5 represents a cyanine-5 monofunctional dye derivative
  • TAM represents a N,N,N' ,N' -tetramethyl-6-carboxyrhodamine derivative
  • each solid line represents a covalent bond
  • R represents either a hydroxy or phosphate terminus
  • Q represents either a hydroxy or phosphate terminus, with the proviso that 'R and Q are different.
  • m is 3, and n is 7.
  • wherein m is 4, and n is 6.
  • m is 5, and n is 5.
  • m is 6, and n is 6.
  • m is 7, and n is 7.
  • This invention also provides a composition of matter comprising the structure shown below:
  • S represents a 1 ', 2 ' -dideoxyribose phosphate moiety
  • m represents an integer greater than 1 and less than 100
  • T represents a thymidine derivative
  • TAM represents a N, N, N' ,N' -tetramethyl-6-carboxyrhodamine derivative
  • each solid line represents a covalent bond
  • R represents either a hydroxy or phosphate terminus
  • Q represents either a hydroxy or phosphate terminus, with the proviso that R and Q are different.
  • m 4
  • This invention also provides a nucleic acid labeled with any of the instant compositions .
  • nucleic acid is DNA. In one embodiment the nucleic acid is RNA.
  • the nucleic acid is DNA/RNA.
  • This invention also provides a method of determining whether a preselected nucleotide residue is present at a predetermined position within a nucleic acid comprising the steps of:
  • This invention further provides a method of determining whether at various predetermined positions within a nucleic acid, a preselected nucleotide residue is present at such position, wherein the preselected nucleotide residue may vary at different predetermined positions which comprises determining whether each preselected nucleotide is present each predetermined position according to the instant method.
  • the presence of a plurality of given nucleotide residues is determined simultaneously .
  • the DNA ligase is Taq DNA ligase.
  • This invention further provides the instant method, wherein the second oligonucleotide has an isolation- permitting moiety affixed thereto, and wherein the method further comprises the steps of isolating the moiety-containing molecules resulting from step (a) and determining the presence therein of ligated first and second oligonucleotides .
  • This invention further provides the instant method, wherein the composition of matter affixed to the first oligonucleotide has a predetermined emission spectrum, and wherein the observation of this emission spectrum is employed to determine the presence of ligated first and second oligonucleotides in step (b) .
  • This invention also provides a method of determining whether a preselected nucleotide residue is present at a predetermined position within a nucleic acid comprising the steps of: (a) contacting the nucleic acid, under hybridizing and DNA polymerization-permitting conditions, with (i) a DNA polymerase, (ii) an oligonucleotide (1) having affixed thereto a composition of matter of claim 1, and (2) having a hydroxyl 3' terminus thereof, wherein the oligonucleotide hybridizes with the 3' region of the nucleic acid molecule flanking the predetermined position, and (iii) a dideoxynucleotide labeled with an isolation- permitting moiety, wherein the labeled dideoxynucleotide is complementary to the given nucleotide residue, with the proviso that upon hybridization of the oligonucleotide with the nucleic acid in the presence of DNA polymerase and the preselected nucleotide
  • This invention further provides a method of determining whether at various predetermined positions within a nucleic acid, a preselected nucleotide residue is present at such position, wherein the preselected nucleotide residue may vary at different predetermined positions which comprises determining whether each preselected nucleotide is present each predetermined position according to the instant method.
  • the DNA polymerase is thermo sequenase .
  • This invention further provides the instant method, ' wherein the dideoxynucleotide is selected from the group consisting of dideoxyadenosine triphosphate, dideoxycytidine triphosphate, dideoxyguanosine triphosphate, dideoxythymidine triphosphate, and dideoxyuridine triphosphate.
  • This invention further provides the instant method, wherein the composition of matter affixed to the oligonucleotide has a predetermined emission spectrum, and wherein the observation of this emission spectrum is employed to determine the presence of polymerization product in step (b) .
  • This invention further provides the instant methods, wherein observing the predetermined emission spectrum is performed using radiation having a wavelength of between 200 and lOOOnm.
  • the radiation has a wavelength of 488 nm.
  • This invention further provides the instant methods, wherein observing the predetermined emission spectrum is performed using radiation having a bandwidth of between 1 and 50nm.
  • the radiation bandwidth is lnm.
  • This invention further provides the instant methods, wherein the isolation-permitting moiety comprises biotin, streptavidin, phenylboronic acid, salicylhydroxamic acid, an antibody or an antigen.
  • This invention further provides the instant methods, wherein the isolation-permitting moiety is attached to the oligonucleotide via a linker molecule.
  • This invention further provides the instant methods, wherein the isolation-permitting moiety is attached to the dideoxynucleotide via a linker molecule.
  • This invention further provides the instant methods, wherein the linker molecule is chemically cleavable.
  • This invention further provides the instant methods, wherein the linker molecule is photocleavable .
  • This invention further provides the instant methods, ' wherein the linker molecule has the structure:
  • the chromophore with high energy absorption is defined as a donor, and the chromophore with lower energy absorption is defined as an acceptor.
  • Fluorescence energy transfer is mediated by a dipole-dipole coupling between the chromophores that results in resonance transfer of excitation energy from an excited donor molecule to an acceptor (Forster, 1965) .
  • F ⁇ rster established that the energy transfer efficiency is proportional to the inverse of the sixth power of the distance between the two chromophores.
  • Fluorescence resonance energy transfer has been used extensively as a spectroscopic ruler for biological structures (Stryer, 1978), and energy transfer-coupled tandem phycobiliprotein conjugates have found wide applications as unique fluorescent labels (Glazer and Stryer, 1983) .
  • a set of polycationic heterodimeric fluorophores that exploit energy transfer and have high affinities for double- stranded DNA were also developed, offering advantages over monomeric fluorophores in multiplex fluorescence labeling applications (Benson et al . , 1993; Rye et al., 1993).
  • the present application discloses how energy transfer and combinatorial concepts can be used to tune the fluorescence emission signature of fluorescent tags for the development of a large number of combina torial fluorescence energy transfer (CFET) tags.
  • CFET combina torial fluorescence energy transfer
  • FAM fluorescence emission maxima for FAM, TAM and Cy5 are selected as examples to construct the CFET tags .
  • spacers Chemical moieties used as spacers are selected to construct various CFET tags aimed at conveniently labeling biomolecules and other targets of interest, monomers are convenient to employ.
  • Other spacer moieties include nucleotides, peptides and 1' 2' -dideoxyribose phosphates.
  • FAM fluorescence emission intensity of the donor
  • TAM acceptor
  • CFET tags 6, 7 and 8 can be generated.
  • Cy5 which acts as the final acceptor
  • CFET tags 9 and 10 can be constructed by manipulating distances "Rl" and "R2". All the CFET tags can be excited with a single laser source and analyzed by simple detectors capable of capturing the emission signatures from each tag. In other embodiments, more than three dyes can be used. Alternatively just single chromophores can be used as long as they have unique fluorescence signatures.
  • the donor and acceptor fluorescent molecules are separated using convenient chemical moieties as spacers to tune the fluorescence signatures of the CFET tags.
  • spacer moieties include nucleotides, dideoxyribose phosphate, and amino acids .
  • the construction of CFET tags involving three or more different dyes is more challenging, since synthetic procedures need to be designed for introducing the individual dye molecules at specific locations on the spacing backbone. As an example,
  • CFET tags involving three dyes can be constructed using oligonucleotides as spacers.
  • SEQ ID NO: 1 was selected as a scaffold to covalently attach FAM, TAM and Cy5.
  • FAM is introduced by using a 6-FAM-dT phosphoramidite
  • TAM is introduced by using TAM-dT (Glen Research, Sterling, VA)
  • a modified T having an amino linker at the C-5 position is incorporated into the oligonucleotide which is then linked to Cy5 - N-Hydroxy succinimide (NHS) ester.
  • the final product is purified by size exclusion chromatography and gel electrophoresis.
  • the fluorescence emission spectrum of F-4-T-6-C displays a fluorescence signature with Cy5 highest, TAM next and FAM lowest; whereas F-7-T-3-C displays a fluorescence signature with FAM highest, TAM next and Cy5 lowest.
  • the two fluorescence signatures are clearly different, and easily discernible by spectroscopic methods. Here the feasibility of the CFET approach involving three different dyes is clearly demonstrated.
  • CFET tags can synthesize broad families of CFET tags. Examples of two synthetic approaches for constructing CFET tags are shown: (1) l',2'- dideoxyribose phosphate monomer can be used as a spacer to separate dyes used for labeling oligonucleotide primers, which can be assembled on a DNA synthesizer; (2) a rigid peptide linker can be used to construct a CFET cassette to label any other molecular targets.
  • a polymer linker formed by 1' , 2' -dideoxyribose phosphates (S) at the 5' end of the desired primer sequence forms a universal spacer for attaching the ET-coupled fluorophores, thereby producing an ET cassette.
  • the 1 ', 2 ' -dideoxyribose phosphates can be introduced using 5 ' -dimethoxytrityl-1 ' , 2 ' - dideoxyribose-3 ' - [ (2-cyanoethyl ) - (N, -diisopropyl ) ] - phosphoramidite (dSpacer CE Phosphoramidite, Glen Research, Sterling, VA) .
  • dSpacer CE Phosphoramidite has previously been used to construct DNA sequencing primers (Ju et al . , 1996). In this CFET tag construction, FAM is used as a common donor.
  • TAM In a CFET tag consisting of two different fluorescent dyes, either TAM or Cy5 can be used as acceptors ; whereas in a CFET tag consisting of three different fluorescent dyes, TAM can also be used as a donor for Cy5.
  • the length of the spacing between each donor/acceptor pair can be changed systematically to achieve the expected fluorescence signatures as shown in Table 1.
  • FAM and TAM can be introduced using phosphoramidite FAM-dT and TAM-dT and Cy5 can be introduced to the modified T carrying an amino linker as described above.
  • spacers are advantageous in several aspects: (i) the spacer will not hybridize to any sequences within the DNA template and therefore false priming is avoided; (ii) the linkage of the spacer maintains the natural nucleic acid phosphate functionality, which avoids possible anomalies in electrophoretic mobility; and (iii) the elimination of the aromatic base groups on the deoxyribose rings in the spacer may reduce the likelihood of fluorescence quenching.
  • Figure 3B shows a general scheme for the construction of CFET- deoxyuridine triphosphate (dUTP) using poly-proline (P) peptide as a spacer. The spacing between each donor/acceptor pair can be changed systematically to achieve the expected fluorescence signatures as shown in Table 1.
  • Figure 4 shows a scheme for the synthesis of CFET-dUTP consisting of Fam, Tam and Cy5. Peptide synthesis procedure using tert- butylcarbonyl (t-Boc) chemistry is employed on a peptide synthesizer to construct the scaffold of the desired molecules.
  • t-Boc tert- butylcarbonyl
  • a modified proline tagged with FAM (Fam- proline) is coupled to glycine, then proline monomers are added, followed by reacting with another modified proline that has a protected primary amino linker (TFA-NH-proline) for the subsequent incorporation of Tam.
  • proline spacer is again added, followed by reacting with the azido-proline for the subsequent incorporation of Cy5.
  • compound 1 in Figure 4 is obtained.
  • Compound 1 reacts with TAM- NHS ester to form compound 2, which will then react with Cy5-phosphine (3) to produce compound 4, which has all the three dyes incorporated.
  • Cy5-phosphine (3) can be synthesized using the modified Staudinger reaction developed by Bertozzi (Saxon and Bertozzi, 2000) . Conversion of compound 4 to an NHS ester produces 5, which is then coupled to Aminoallyl (AA) - dUTP (Sigma) to generate the final product CFET-dUTP . By varying the number of proline spacers between Fam and Tam, and between Tam and Cy5, a library of CFET- dUTPs with unique fluorescence signatures can be developed. The intermediates 2, 4, 5, and the final products can be purified by high pressure liquid chromatography (HPLC) , size exclusion chromatography and gel electrophoresis.
  • HPLC high pressure liquid chromatography
  • Sophisticated techniques have enabled large-scale dissection of genomes. For instance, the development of cloning vectors which can maintain and reproduce large stretches of DNA (up to a million bases) has resulted in clone libraries which span most of the chromosomes from end to end for many of the highly studied organisms including humans - so-called physical maps. Recognizing sequence markers that differ from one individual to another across the human genome has permitted them to be followed in families that harbor genetic diseases. If a marker cosegregates with the disease phenotype, one can be assured that the marker is in the vicinity of the gene responsible for that disease.
  • DNA sequencing serves as a good example for evaluating the impact of this technology. Although the ability to obtain DNA sequences originated in the late 1970 's with the development of the chemical cleavage approach of Maxa and Gilbert (1977) and the dideoxynucleotide terminator approach of Sanger et al . (1977), it was the latter that was most amenable to automation and fluorescent labeling strategies.
  • Ligase chain reaction is a procedure for genetic mutation analysis using ligase and a pair of oligonucleotides (Eggerding, 1995; Wu and Wallace, 1989; Landegren et al . , 1988). Briefly, it is based on the fact that two adjacent oligonucleotides can only be ligated if the adjoining bases are complementary to the template strand. If there is a single base difference within two bases of the join site, ligation will not occur. Pairs of oligonucleotides are designed spanning the ligation site on the template DNA, including one harboring either the wild-type or mutated base.
  • one of the oligonucleotides is radiolabeled at the phosphate group at its 5' end.
  • ligase chain reaction which involves multiple rounds of denaturing, primer annealing- and ligation, one can separate the products from the substrates on polyacrylamide gels.
  • the procedure can be modified using single stranded DNA template as shown in Figure 8 for testing using the CFET tags.
  • Primer pairs are generated surrounding a base that can be mutated.
  • the template may contain a T (wild-type, wt) or C (mutated, ut) at the relevant position.
  • the wt primers are complementary to the wt template at every position.
  • the primer on the right side of Figure 8A is labeled with CFET tag 1 to yield a specific fluorescent signature.
  • the mutation-specific primer two bases longer than its wild-type analog, is complementary to every position of the mutated template.
  • This primer is labeled with CFET tag 2 displaying another unique fluorescent signature.
  • a common 20 base pair primer will be used on the other side of the ligation site. In cases where ligation does not occur, because a wild-type oligonucleotide was used with a mutated template sequence, or a mutated oligonucleotide was used with a wild-type template sequence, the only fluorescent band on the acrylamide gel will be the size of the tagged primer.
  • the multiplex set of oligonucleotides that contain the potentially mutated position can be 5' -end labeled, each with a specific CFET tag. For example, one can test four different mutation sites using eight distinct CFET tags.
  • воднру ⁇ е CFET tags 1, 2, 3, 4, 5, 6, 9, and 10 of Table 1
  • S 1' , 2 ' -dideoxysugar phosphate
  • FAM is used as a common donor
  • TAM and/or Cy5 as acceptors.
  • the length of the spacing between each donor/acceptor pair, (S) m and (S) n can be changed systematically to achieve the expected fluorescence signatures as depicted in Table 1.
  • FAM and TAM can be introduced using FAM-dT and TAM-dT phosphoramidites and Cy5 can be introduced to the modified T carrying an amino linker as described above .
  • the system can be tested, for example, by synthesizing single stranded DNA templates mimicking known single base mutations in exon 20 of the retinoblastoma susceptibility (RBI) gene (Schubert et al . 1994, Lohmann 1999).
  • the sequences of two sets of synthetic templates (wt and mut) which can be used in the analysis are shown in Table 3.
  • the sequence of the potential mutation positions is shown in bold- face as "A', "C", "G” and "T”.
  • Primer sets 1 and 2 in Table 2 are used for the testing of both wild type and mutated base positions of Template A, respectively; while primer sets 3 and 4 are for testing both wild type and mutated base positions of Template B, respectively.
  • the primers surrounding each "mutated" position can be designed to be a unique length as shown in Figure 9.
  • the two CFET labeled oligonucleotides (one for the wild-type gene and one for the mutated gene) surrounding mutation position 1 are 20 and 22 bases long, respectively, and the unlabeled common primer is 20 bases long. Any resulting ligation product will be either 40 or 42 bases long.
  • 24 and 26 base labeled oligonucleotides can be constructed, as well as a different 20 base common primer, leading to ligation products of either 44 or 46 bases.
  • More primers can, of course, be generated by making the sizing increment one base instead of two bases for each different mutation, or creating a second set of labeled primers whose ligation products run between 80 and 98 base pairs, between 120 and 138 base pairs, etc. Since single base pair resolution up to the length of ⁇ 400 bp DNA fragments is easily achieved in polyacrylamide gel electrophoresis, the ligated products can be readily resolved in such standard fluorescent gel systems. Furthermore, the advantage of being able to clearly distinguish the products based on their fluorescent signatures, as well as size, makes this assay extremely powerful. Expected gel electrophoresis results for this multiplex testing system are shown on the right side of Figure 9. Here, template collection 1 is seen to contain only wt sequences. In contrast, template pool 2 contains one template with a mutation at position 2 and a heterozygote genotype at position 4.
  • Primer 1L 3 ' -ttaaaaagaataagggtgtc-5 '
  • Primer 2L 3 ' -acatagccgatcggatagag-5 '
  • Primer 2R mut 3 ' -Accgatttatgtgaaacacttgcgga-5 ' -CFET4
  • Primer 3L 3 ' -cggaagacagactcgtgggt-5 ' (SEQ ID NO: 8)
  • Primer 4L 3 ' -atagtagacctgggaaaagg-5 '
  • Template B 5 ' -tacactttgtgaacgccttctgtctgagcacccaGaattagaacatatca tctggacccttttccAgcacaccctgcagaatgagtatgaactcatgaga-3 ' (wild type) (SEQ ID NO: 16)
  • Probes can be generated using a random primed labeling method to incorporate CFET-dUTP into chromosome-specific DNA molecules or cos ids disposed along the length of a given chromosome. Metaphase spreads of fresh cells or deparaffinized material can be prepared by standard methodologies, and the tagged probes can be hybridized to the chromosomes. Bulky ET dyes consisting of two individual fluorescent molecules, as well as dyes with a long linker, have been attached to deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs) which have been shown to be good substrates for DNA polymerase (Rosenblum et al. 1997, Zhu et al . 1994).
  • dNTPs deoxynucleotides
  • ddNTPs dideoxynucleotides
  • the CFET-dUTP should be able to be incorporated into the growing strand by the polymerase reaction.
  • the ratio of regular deoxythymine triphosphate (dTTP) and CFET-dUTP can be adjusted, so that only a small portion of CFET-dUTP will be incorporated into the growing chain, just enough to be detected by the optical method.
  • Numerical and structural chromosome rearrangements are a major cause of human mortality and morbidity. Aneuploidy of whole chromosomes accounts for at least 50% of early embryonic lethality, and also leads to severe patterns of congenital malformation such as Down syndrome. Segmental aneuploidies due to deletions and duplications also lead to malformation syndromes, as well as being associated with many types of cancer.
  • M-FISH and Spectral Karyotyping use a combinatorial approach of five dyes to "paint" all 23 pairs of human chromosomes so they can be distinguished using computerized image software (Schrock et al . 1996, Speicher et al . 1996).
  • these established techniques require careful mixing of dyes in controlled ratios. Quality control is often a problem, and the commercially available probes are very expensive.
  • CFET Tags are expected to have a substantial advantage over currently available dye sets. It should be possible to generate a larger number of CFET tag sets, reducing the need for a combinatorial approach. Quality control is also likely to be easier, since each probe needs to be labeled with only one tag, and probe sets can be mixed in equal quantities to produce multicolor FISH reagents.
  • CFET Tags for example could be used both for the detection of aneuploidy in interphase nuclei, and for the detection of submicroscopic chromosomal deletions and amplifications.
  • a set of eight different CFET tag labeled probes can be prepared, each specific for one of the chromosomes most commonly involved in aneuploidy in either embryonic losses or birth defects (chromosomes 13, 15, 16, 18, 21, 22, X and Y) .
  • FIG. 10 A schematic of a procedure for comprehensive chromosome-wide analysis for gain or loss of genetic material is shown in Figure 10.
  • eight probes each labeled with a CFET-dUTP that emits a unique fluorescence signature are hybridized along a chromosome in eight separate locations.
  • the normal chromosome A will display eight unique fluorescence signatures of each probe in a defined order.
  • a loss of fluorescence signature "2" in chromosome B will indicate the deletion of the complementary sequence of probe 2.
  • the appearance of two signatures of "3" will indicate the expansion of the complementary sequences for probe 3.
  • the CFET tags with unique fluorescence signatures which are disclosed in the present application will have utility in other applications involving multi component analysis in addition to those disclosed above. Additional applications include, but are not limited to, multiplex assays including binding assays and immuno assays, detection of microbial pathogens, monitoring multiple biomolecular reactions, screening of drugs or compounds, epitope mapping, allergy screening, and use with organic compounds and in material science. For example, multiple reactions or interactions can be measured simultaneously, where multiple CFET tags, each with a different fluorescence signature, are used to label the different reactants which could include, for example, antibodies, antigens, ligands, or substrates. Examples include antibody-antigen and receptor-ligand binding. In further examples, different reactants can be coupled to microspheres . VI . CFET Tags Used in Ligation Assay to Identify Multiple Single Nucleotide Polymorphisms .
  • the CFET tags were applied to an oligonucleotide ligation assay (Landegren, 1988) coupled with solid phase purification to detect genetic mutations on exon 20 of the tumor suppressor retinoblastoma (RBI) gene.
  • RBI tumor suppressor retinoblastoma
  • Two 20 base-pair oligonucleotides one labeled with a CFET tag at the 5' end and the other labeled with a biotin at the 3' end and a monophosphate (P) group at the 5' end, are hybridized to the target DNA template such that the 3' end of the CFET-labeled oligonucleotide is positioned next to the 5' end of the biotinylated oligonucleotide.
  • Taq DNA ligase joins the two juxtaposed oligonucleotides in a head- to-tail fashion by forming a phosphodiester bond, provided that the nucleotides at the ligating junction of the two oligonucleotides are correctly base-paired with the template (Barany, 1991) .
  • no ligation reaction occurs when there is a mismatch between the 3' end of the CFET-labeled probe (nucleotides A and C, Fig. 11) and the SNP site (nucleotides T and G, Fig. 11) on the target template.
  • the CFET-labeled ligation products (40 base-pair) are immobilized to streptavidin-coated magnetic beads while the other components are washed away.
  • the ligation products are then cleaved from the magnetic beads by denaturing the biotin-streptavidin interaction with formamide and analyzed with a three-color fluorescence CAE system.
  • the CFET-labeled ligation products are unambiguously detected due to their distinct mobility and unique fluorescence signatures in the electropherogram, see Figure 12.
  • two CFET tags with different fluorescence signature and electrophoretic mobility are used to label the oligonucleotides corresponding to each allele.
  • the unique fluorescence signatures in the electropherogram thus identify each of the corresponding SNPs .
  • the solid phase procedure completely eliminates the unligated CFET-labeled oligonucleotide. Although the unligated 20 base-pair biotinylated oligonucleotides are also captured by the magnetic beads, they do not produce fluorescence signals due to the absence of CFET tags.
  • the CFET tag library in this application detects multiple SNPs on the target DNA template simultaneously.
  • Exon 20 of the tumor suppressor RBI gene was selected as a model system to test the utility of the CFET tags.
  • Several SNPs within a region of 200 base pairs in the RBI gene have been found, which are well suited for evaluating a genetic mutation analysis system.
  • Six ligation reactions were carried out separately using six different CFET tags on synthetic templates mimicking exon 20 of the RBI gene where multiple SNPs (six nucleotide variations) are located.
  • the ligation products were combined in a single tube and analyzed with a three-color CAE system, resulting in the simultaneous detection of six nucleotide variations by the unique fluorescence signatures of the CFET-labeled ligation products (see Figure 12A) .
  • the unique fluorescence signatures were spatially resolved in the electropherogram as a result of the different mobility of the CFET-labeled ligation products.
  • both CFET-1 (FAM) and CFET-6 (F-10-Cy5) detect homozygous SNPs (T/T) .
  • CFET-3 (F-9-T) and CFET-4 (F-13-T) clearly distinguish a mimic of RBI gene mutation R661W (amino acid change from arginine to tryptophan due to mutation in codon 661) by detecting both the wild type (C) and " the mutation (T) .
  • CFET-7 (F-4-T-6- Cy5) and CFET-8 (F-7-T-7-Cy5) identify another mutation Q685P (amino acid change from glutamine to proline due to mutation in codon 685) with heterozygous genotype (A/C) .
  • CFET-1, 3 and 7 CFET-labeled oligonucleotide probes
  • biotinylated oligonucleotides to identify three SNPs using a PCR product amplified from exon 20 of the RBI gene from patient genomic DNA.
  • the ligation reactions were performed in a single tube and the reaction products were loaded onto a three-color CAE system.
  • T Three individual homozygous SNPs (T, C and A) , that were verified by DNA sequencing, were unambiguously identified by the three distinct fluorescence signatures from the CFET tags (figure 12B) : T (FAM, CFET-1), C (F-9-T, CFET-3) and A (F-4-T-6-Cy5, CFET-7) .
  • T FAM, CFET-1
  • C F-9-T, CFET-3
  • A F-4-T-6-Cy5, CFET-7 .
  • the approach described here can detect both heterozygotes and homozygotes unambiguously because of the unique CFET fluorescence signature and mobility in the electropherogram.
  • isolation-permitting moieties besides biotin may be employed such as phenylboronic acid. Attachment of the moieties via cleavable linker molecules enhances this still further.
  • Single base extension for each dye-labeled primer was done by mixing 0.5 to 1 pmol of the primers with 1 pmol of template, followed by adding 2 ⁇ l of ther o sequenase 10X reaction buffer (260 mM Tris-HCl, 65 mM MgCl 2 , pH 9.5, Amersham Pharmacia Biotech, Piscataway, NJ) , 5 ⁇ l of water, 1 pmol of biotinylated dideoxynucleoside triphosphates (Biotin-11-ddNTP, NEN, Boston, MA) and 1 unit of thermo sequenase in 20 mM Tris-HCl, pH 8.5, 50% glycerol, 0.1 mM ethylenediamine tetraacetic acid (EDTA) , 0.5% TweenTM- 20 (v/v) , 0.5% NonidetTM P-40 (v/v) , lmM dithiothreitol (DTT) , 100 m
  • reaction mixture was incubated at 54 °C for 30 sec for single base extension.
  • extension of the primers are initiated by ddCTP-Biotin (for primer 1) and ddGTP-Biotin (for primer 2) in the presence of DNA polymerase if there is a match between the 3' end of the primer and the template (X and Y for primer 1; X' and Y' for primer 2) .
  • the extension products are isolated using streptavidin-coated magnetic beads. Upon denaturing, washing and releasing from the beads, the extension products are loaded onto an electrophoresis system and the resulting fluorescence signatures from the electropherogram identify each of the unique SNPs .
  • the CFET-labeled oligonucleotides, DNA polymerase and biotinylated dideoxynucleotides form a high fidelity SNP detection system in which the base at the 3' end of the oligonucleotides dictates its extension by incorporating a specific biotinylated dideoxynucleotide.
  • the CFET tags used were F, F-9-T and F-13-T. Their unique fluorescence signatures are shown in Figures 14 and 15
  • isolation-permitting moieties such as phenylboronic acid, antigens or antibodies may be employed in place of the biotin. Attachment of the moieties via cleavable linker molecules enhances this still further.
  • the throughput of the multiplex analyses offered by the use of the CFET tags can be increased by performing the analyses in the high throughput chamber illustrated in figure 16.
  • CFET tags can be used in combination with single chromophore/fluorophore tags and tags with multiple chromophores/fluorophores where no FET occurs.
  • fluorophores could be quantum dots, luminescent molecules of fluorescent dyes.
  • each tag could be used to detect a different SNP using the exemplified assays.
  • Template-directed dye- terminator incorporation (TDI) assay a homogeneous DNA diagnostic method based on fluorescence resonance energy transfer Nucl . Acids . Res . 25: 347-353.

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