EP0833936A4 - Nouvelles amorces marquees au colorant, ribonucleotides, desoxyribonucleotides et didesoxyribonucleotides servant a effectuer une analyse automatique d'adn et a mettre en application des methodes de detection et d'amplification homogenes - Google Patents

Nouvelles amorces marquees au colorant, ribonucleotides, desoxyribonucleotides et didesoxyribonucleotides servant a effectuer une analyse automatique d'adn et a mettre en application des methodes de detection et d'amplification homogenes

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Publication number
EP0833936A4
EP0833936A4 EP96921749A EP96921749A EP0833936A4 EP 0833936 A4 EP0833936 A4 EP 0833936A4 EP 96921749 A EP96921749 A EP 96921749A EP 96921749 A EP96921749 A EP 96921749A EP 0833936 A4 EP0833936 A4 EP 0833936A4
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European Patent Office
Prior art keywords
bodipy
fluorophore
fluorophores
labeled
polynucleotides
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EP96921749A
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German (de)
English (en)
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EP0833936A1 (fr
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Michael L Metzker
Richard A Gibbs
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Baylor College of Medicine
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Baylor College of Medicine
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Priority claimed from US08/494,216 external-priority patent/US5614386A/en
Priority claimed from US08/540,228 external-priority patent/US5861287A/en
Priority claimed from US08/553,936 external-priority patent/US5728529A/en
Priority claimed from US08/612,036 external-priority patent/US5994063A/en
Application filed by Baylor College of Medicine filed Critical Baylor College of Medicine
Publication of EP0833936A1 publication Critical patent/EP0833936A1/fr
Publication of EP0833936A4 publication Critical patent/EP0833936A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • This invention relates generally to methods for the use of a class of substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY ® fluorophore) compounds for improved DNA sequencing by chemical cleavage and by hybridization, labelling of DNA fragments for genetic analysis, improved DNA sequencing by the chain termination method of BODIPY ® fluorophore
  • Native DNA consists of two linear polymers, or strands, of nucleotides. Each strand is a chain of nucleosides linked by phosphodiester bonds. The two strands are held together in an antiparallel orientation by hydrogen bonds between complementary bases of the nucleotides of the two strands: deoxyadenosine triphosphate (A) pairs with thymidine triphosphate (T) and deoxyguanosine triphosphate (G) pairs with deoxycytidine triphosphate (C).
  • A deoxyadenosine triphosphate
  • T thymidine triphosphate
  • G deoxyguanosine triphosphate
  • RNA ribonucleic acid
  • nucleic acid sequencing allows dissection of animal, plant and viral genomes into discrete genes with defined chemical structure. Since the function of a biological molecule is determined by its structure, defining the structure of a gene is crucial to the eventual useful manipulation of this basic unit of hereditary information. Once genes are isolated and characterized, they can be modified to produce desired changes in their structure that allow the production of gene products-proteins-with different properties than those possessed by the original gene products.
  • nucleic acid sequencing methods involved parallel developments in a variety of techniques.
  • the key development was the introduction of methods of generating sets of fragments of cloned, purified DNA that contain, in their collection of lengths, the information necessary to define the sequence of the nucleotides comprising the parent DNA molecules.
  • the method developed by Sanger is referred to as the dideoxy chain termination method.
  • a DNA segment is cloned into a single-stranded DNA phage such as M13.
  • M13 DNA phage
  • These phage DNAs can serve as templates for the primed synthesis of the complementary strand by conventional DNA polymerases.
  • the primer is either a synthetic oligonucleotide or a restriction fragment isolated from the parental recombinant DNA that hybridizes specifically to a region of the M13 vector near the 3' end of the cloned insert.
  • the primed synthesis is carried out in the presence of enough of the dideoxy analog of one of the four possible deoxynucleotides so that the growing chains are randomly terminated by the incorporation of these "deadend" nucleotides.
  • the relative concentration of dideoxy to deoxy forms is adjusted to give a spread of termination events corresponding to all the possible chain lengths that can be resolved by gel electrophoresis.
  • the products from each of the four primed synthesis reactions are loaded into individual lanes and are separated by polyacrylamide gel electrophoresis. Radioactive label incorporated in the growing chains are used to develop an autoradiogram image of the pattern of the DNA in each electrophoresis lane.
  • the sequence of the deoxynucleotides in the cloned DNA is determined from an examination of the pattern of bands in the four lanes. Because the products from each of the four synthesis reactions must be run on separate gel lanes, there are problems with comparing band mobilities in the different lanes.
  • fragments having different terminating bases can be labeled with different fluorescent dyes, which are attached either to a primer for dye-primer sequencing in which the fluorescent dyes are attached to the 5* end ofthe primers, e.g., Smith et al.
  • a fluorescence detector then can be used to detect the fluorophore-labeled DNA fragments.
  • the four different dideoxy-terminated samples can be run in four separate lanes or, if labeled differentially, in the same lane.
  • the method of Fung, et al., U.S. Patent No. 4,855,225 uses a set of four chromophores or fluorophores with different absorption or fluorescent maxima. Each of these tags is coupled chemically to the primer used to initiate the synthesis ofthe fragment strands. In turn, each tagged primer is then paired with one of the dideoxynucleotides and used in the primed synthesis reaction with conventional DNA polymerases. The labeled fragments are then combined and loaded onto the same gel column for electrophoretic separation. Base sequence is determined by analyzing the fluorescent signals emitted by the fragments as they pass a stationary detector during the separation process. Obtaining a set of dyes to label the different fragments is a major difficulty in automated DNA sequencing systems.
  • Another difficulty with obtaining an appropriate set of dyes is that when several fluorescent dyes are used concurrently, excitation becomes difficult because the absorption bands of the dyes are often widely separated. The most efficient excitation occurs when each dye is illuminated at the wavelength corresponding to its absorption band maximum. Thus, one often is forced to compromise between the sensitivity of the detection system and the increased cost of providing separate excitation sources for each dye.
  • the number of differently sized fragments in a single column of a gel is greater than a few hundred, the physiochemical properties of the dyes and the means by which they are linked to the fragments become critical because the charge, molecular weight, and conformation of the dyes and linkers must not effect adversely the electrophoretic mobilities of closely-sized fragments.
  • DNA sequencing would be advanced significantly by the availability of new sets of fluorescent dyes which (1) are physiochemically similar, (2) permit detection of spatially overlapping target substances, such as closely spaced bands of DNA on a gel, (3) extend the number of bases that can be determined on a single gel column by current methods of automated DNA sequencing, (4) are amenable for use with a wide range of preparative and manipulative techniques, and (5) otherwise satisfy the numerous requirements listed above. See, Bergot, et al. (cited above).
  • Patent No. 5,171,534 Thus, the set of discriminating fluorophores described in the literature is small, and the search for improved, alternative dyes has been difficult at best.
  • fluorescein and its derivative dyes labeled in DNA sequencing reactions have different gel mobilities in comparison to rhodamine and its derivative dyes labeled in DNA sequencing reactions. Fluorescein and its derivative dye-labeled reactions typically move through the gel faster (sometimes greater than one base position) than rhodamine and its derivative dye-labeled reactions.
  • each fluorophore is coupled to the primer via different linker arm lengths.
  • Both fluoresceins are coupled to the primer using a two-carbon amino linker arm while both rhodamines are coupled to the primer using six- carbon amino linker arm.
  • Mobility correction software is required additionally to generate properly spaced DNA termination fragments.
  • custom sequencing primers These primers refer to any oligonucleotide sequence that can act as a suitable DNA sequencing primer.
  • a 5'-leader sequence (5'-CAGGA) must be coupled to the primer and custom sequencing primers must use the M13RP1 mobility correction software to generate properly-spaced DNA termination fragments.
  • the leader sequence is the first five bases of the reverse M13RP1 sequencing primer.
  • M13RP1 is the mobility software file used to generate properly spaced DNA termination fragments for the reverse sequencing primer.
  • PCR polymerase chain reaction
  • the term "polymerase chain reaction” or "PCR” refers generally to the procedure involving: (1) treating extracted DNA to form single- stranded complementary strands; (2) adding a pair of oligonucleotide primers, wherein one primer of the pair is substantially complementary to part of the sequence in the sense strand and the other primer of each pair is substantially complementary to a different part of the same sequence in the complementary antisense strand; (3) annealing the paired primers to the complementary sequence; (4) simultaneously extending the annealed primers from a 3' terminus of each primer to synthesize an extension product complementary to the strands annealed to each primer wherein said extension products after separation from the complement serve as templates for the synthesis of an extension product for the other primer of each pair; (5) separating said extension products from said
  • Detection methods generally employed in standard PCR techniques use a labeled probe with the amplified DNA in a hybridization assay.
  • PCR FLP fragment length polymorphism
  • ASO allele-specific oligonucleotide
  • ASO allele-specific oligonucleotide
  • the standard PCR technique operates by replicating a DNA sequence positioned between two primers, providing as the major product of the reaction a DNA sequence of discrete length terminating with the primer at the 5' end of each strand.
  • insertions and deletions between the primers result in product sequence of different lengths, which can be detected by sizing the product in PCR-FLP.
  • ASO hybridization the amplified
  • DNA is fixed to a nylon filter (by, for example UN irradiation) in a series of "dot blots", then allowed to hybridize with an oligonucleotide probe labeled with HRP under stringent conditions. After washing, tetrametholbenzidine (TMB) and hydrogen peroxide are added: HRP oxidizes the hydrogen peroxide, which in turn oxidizes the TMB to a blue precipitate, indicating hybridized probe.
  • TMB tetrametholbenzidine
  • HRP oxidizes the hydrogen peroxide, which in turn oxidizes the TMB to a blue precipitate, indicating hybridized probe.
  • the 5' - 3' exonuclease activity of Taq polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe.
  • the assay is sensitive and specific and is a significant improvement over more cumbersome detection methods. A version of this assay is also described in Gelfand et al., in U.S. Patent No. 5,210,015.
  • U.S. Pat. No. 5,491,063 to Fisher, et al. provides a Taqman-type assay.
  • the method of Fisher et al. provides a reaction that results in the cleavage of single-stranded oligonucleotide probes labeled with a light-emitting label wherein the reaction is carried out in the presence of a DNA binding compound that interacts with the label to modify the light emission of the label.
  • the method utilizes the change in light emission of the labeled probe that results from degradation of the probe.
  • the methods are applicable in general to assays that utilize a reaction that results in cleavage of oligonucleotide probes, and in particular, to homogeneous amplification/detection assays where hybridized probe is cleaved concomitant with primer extension.
  • a homogeneous amplification/detection assay is provided which allows the simultaneous detection of the accumulation of amplified target and the sequence-specific detection of the target sequence.
  • BODIPY® fluorophores A new class of dyes, BODIPY® fluorophores, has been recently described. See, Haugland, et al., Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals, pp. 24-32, and U.S. Patent No. 4,774,339.
  • the parent heterocyclic molecule of the BODIPY® fluorophores is a dipyrrometheneboron difluoride compound and which is modified to create a broad class of spectrally-discriminating fluorophores, see Figure 1.
  • the conventional naming of these dyes is BODIPY® followed by their approximate absorption/emission maxima, e.g., BODIPY ® 530/550.
  • the present invention provides for BODIPY® fluorophores for methods for DNA sequencing by chemical cleavage, hybridization, chain termination, for genetic analysis and for performing the Taqman assay.
  • additional prior art techniques include the following:
  • U.S. Patent No. 4,318,846 to Khanna et al. is drawn to diether symmetrically-substituted fluoresceins having at least one anionic group and a linking functionality. Depending on the site of substitution, the compounds can be used as fluorescers absorbing at wavelengths in excess of 500 nm. The compounds can be used as labels in fluorescent immunoassays.
  • U.S. Patent No. 4,811,218 to Hunkapiller et al. is drawn to a real ⁇ time, automated nucleic acid sequencing apparatus which permits more than one clone to be sequenced at the same time.
  • U.S. Patent No. 4,855,225 to Fung et al. is drawn to a method for detecting up to four sets of oligonucleotides that have been differentially- labeled with fluorophores, two of the sets with substituted fluoresceins and two sets with substituted rhodamines, and separated by gel electrophoresis.
  • U.S. Patent No. 5,366,860 to Bergot et al. is drawn to a method for detecting up to four sets of oligonucleotides that have been differentially- labeled with fluorophores, all rhodamines with different substitutions, and separated by gel electrophoresis.
  • U.S. Patent No. 5,188,934 to Menchen, et al. is drawn to a method for detecting up to four sets of oligonucleotides that have been differentially-labeled with fluorophores, all fluoresceins with different substitutions, and separated by gel electrophoresis.
  • U.S. Patent No. 5,171,534 to Smith et al. describes a system for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations.
  • the system comprises a source of chromophore or fluorescent tagged DNA fragments, a zone for contacting an electrophoresis gel, means for introducing said tagged DNA fragments to said zone and photometric means for monitoring the tagged DNA fragments as they move through the gel.
  • U.S. Patent No. 5,366,603 is drawn to automatic DNA sequencing wherein the DNA is marked with near infrared fluorescent dyes.
  • U.S. Patent No. 5,241,060 to Englehardt, et al. is drawn to labeled nucleotides and polynucleotides with the formula PM-SM-BASE-Sig, where PM is a phosphate moiety, SM is a sugar moiety, BASE is a purine, pyrimidine or 7-deazapurine moiety, and Sig is a detectable moiety that is covalently attached to the BASE entity at a position other than the C 5 position when BASE is a pyrimidine, at a position other than the C 8 position when BASE is a purine and at a position other than the C 7 position when BASE is a 7-deazapurine.
  • U.S. Patent No. 4,755,458 to Rabbani, et al. is drawn to compositions for detecting the presence of a nucleotides sequence of interest.
  • the composition includes a first poiynucleotide molecule is substantially complementary to and capable of hybridizing with a specific sequence of interest and which is labeled with a first detectable marker; a second poiynucleotide molecule is not substantially complementary to and is not capable of hybridizing with the specific sequence of interest and is labeled with the same, first detectable marker; and a third poiynucleotide molecule that is substantially complementary to or substantially identical to the second poiynucleotide but is unlabeled or labeled with a second detectable marker.
  • U.S. Patent No. 5,274,113 to Kang, et al. is drawn to derivatives of dipyrrometheneboron difluoride fluorescent dyes that can be attached to nucleic acids, proteins, carbohydrates and other biologically-derived materials.
  • the compounds of Kang, et al. show various functional groups for attachment of the dipyrrometheneboron difluoride fluorescent dyes to the biologically-derived materials.
  • BODIPY® fluorophores have improved spectral characteristics compared to conventional fluorescein and rhodamine dyes.
  • the BODIPY® fluorophores have narrower band width, insensitivity to solvent or pH, and improved photostability.
  • the use of BODIPY® fluorophores leads to improved DNA sequencing or analysis of DNA fragments in any method where electrophoresis of BODIPY®-labeled DNA is required. It is an object of the present invention to provide methods for the use of a class of dyes particularly suited for DNA sequencing.
  • BODIPY® fluorophores for any method of DNA sequencing in which poiynucleotide products of the sequencing reaction are 5'-end- labelled with said BODIPY® fluorophores. It is a further object of the present invention to provide methods for the use of BODIPY® fluorophores which have been chemically-modified so that a BODIPY ® fluorophore can be used to replace a prior art 5'-end- labelled fluorophore in DNA sequencing and conventional software may be used. BODIPY® fluorophores can be used in one out of the four reactions, two out of the four reactions or three out of the four reactions or in all four reactions.
  • a particular object of the present invention is to provide methods for the use of BODIPY® fluorophores for automated DNA sequencing which, since the particular BODIPY® fluorophores alter the mobility of the corresponding termination products in the same way, nullifies the need for software correction to generate evenly-spaced DNA sequences.
  • An additional object of the present invention is to provide methods for the use of BODIPY® fluorophores for DNA sequencing wherein the BODIPY® fluorophore is attached at the 5' end of the poiynucleotide product of the sequencing reaction and at the 3' end or at one or more internal positions of the products of the sequencing reaction.
  • a method for analysis of DNA fragments wherein said DNA fragments are labelled with at least one BODIPY® fluorophore.
  • a method for distinguishing polynucleotides having different 3'-terminal dideoxynucleotides in any method of DNA sequencing requiring electrophoresis of the products of the sequencing reactions comprising the steps of: forming a mixture ofa first, a second, a third, and a fourth class of polynucleotides, each poiynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled with a first fluorophore; each poiynucleotide in the second class having a 3'-terminal dideoxycytidine and being labeled with a second fluorophore; each poiynucleo
  • BODIPY® fluorophores for DNA sequencing wherein the BODIPY ® fluorophore is attached to a nucleotide at a 3' BODIPY® position.
  • a method for distinguishing polynucleotides having different 3'- terminal dideoxyribonucleotides in any method of chain termination DNA sequencing comprising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each poiynucleotide in the first class having a 3'-terminal dideoxyadenosine triphosphate, said 3'-terminal dideoxyadenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-l-propynyl linker, said linker then attached to a BODIPY® linker at a 3 position of a first BODIPY ® fluorophore that contains at least one reactive functional group; each poiynucleotide in the second class having a 3'-terminal dideoxycytidine tri
  • a method for distinguishing polynucleotides having different ribonucleotides in any method of labelling polynucleotides by enzymatic incorporation comprising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each poiynucleotide in the first class having an adenosine triphosphate, said adenosine triphosphate being attached at the 7 position of the 7- deazapurine to a 3-amino-l-propynyl linker, said linker then attached to a BODIPY ® linker at a 3 position of a first BODIPY® fluorophore that contains at least one reactive functional group; each poiynucleotide in the second class having a cytidine triphosphate, said cytidine triphosphate being attached at the 5 position ofthe pyrim
  • a method for a method for distinguishing polynucleotides having different deoxyribonucleotides in any method of labelling polynucleotides by enzymatic incorporation comprising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each poiynucleotide in the first class having a deoxyadenosine triphosphate, said deoxyadenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-l-propynyl linker, said linker then attached to a BODIPY® linker at a 3 position of a first BODIPY® fluorophore that contains at least one reactive functional group; each poiynucleotide in the second class having a deoxycytidine triphosphate, said deoxycytidine triphosphate being
  • FIG. 1 Chemical structures of several DNA sequencing fluorophores are shown.
  • BET primers double dye-labeled primers FET-3 and BET-3 are shown. Since different protecting groups block the linker arm amines, BET primers were first labeled internally with BODIPY" 503/512. Following removal ofthe monomethoxytrityl group, BET primers were end-labeled with the BODIPY * dye set.
  • the (CH 2 ) n for BET primers correspond to (CH 2 ) 3 for BODIPY * 581/591 and (CH 2 ) ⁇ for BODIPY * 503/512, BODIPY * 523/547, and BODIPY * 564/570 dyes.
  • Figure 3 Depicts the results of a dye-labeled substitution experiment.
  • DNA sequencing reactions were generated by solid-phase Bst sequencing. The region shown corresponds to approximately 230 to 240 bases (Blue), 160 to 170 bases (Green), 290 to 300 bases (Black), and 200 to 210 bases (Red) in the sequencing read.
  • 373A raw files were analyzed by the ABI sequencing analysis version 2.1.0 software program using the
  • ABI50 standard base caller with the M13RP1 mobility correction file.
  • the 1 max (parentheses) for dye-primers was determined using a Model F-4010 fluorescence spectrophotometer (Hitachi, Ltd) in IX TBE buffer (0.089 M Tris-borate, 0.002 M Na ⁇ EDTA) containing 7 M urea. Signal strength was measured using a 373A sequencer (373A) or using a fluorescence spectrophotometer (Spec). 373A measurements were determined by M13 cycling sequencing reactions of four different molecular clones. The relative intensity values were determined by normalizing the BODIPY * dye signal to the remaining dye signals and comparing it to its normalized conventional dye signal.
  • Figure 4 Demonstrates that BODIPY® dye-labeled primers do not require gel mobility correction.
  • -21M13 primers and BODIPY ® primers were used to sequence two different M13 clones by cycle sequencing.
  • 21M13 primers contain FAM-"C", JOE-"A", TAMRA-"G", AND ROX-"T" dye labels and
  • BODIPY® primers contain BODIPY® 503/512-"C", BODIPY ® 530/550- ⁇ ", BODIPY® 564/570-"G", BODIPY® 581/591-”T” dye labels.
  • Arrows above the sequence chromatograms highlight base calling errors, and the approximate base regions from the primer peak are listed.
  • FIG. 5 A general synthetic scheme for end labeling (Route I) and internal labeling (Route II) BODIPY® phosphoramidites is depicted. For specific BODIPY® chemical structures, see Figure 1.
  • Figure 6 Depicts normalized emission spectra of four conventional dye-primers and BODIPY® dye-primers.
  • FIG. 8 (A) Double dye-labeled primers. Since different protecting groups block the linker arm amines, BODIPY energy transfer (BET) primers were first labeled internally with BODIPY 503/512,
  • BODIPY 523/547 or BODIPY 530/550 After removal of the monomethoxytrityl group, BET primers were end-labeled with the BODIPY dye set.
  • B.A. Larder "Reverse Transcriptase", A.M. Skalka and S.P. Goff, Eds., pp. 205-222 (Cold Spring Harbor Laboratory Press, 1993).
  • BET probe labeled with BODIPY 503/512 is specific for the wild-type sequence and BET probe labeled with BODIPY 523/547 is specific for the drug resistance sequence.
  • BODIPY® shall refer to a class of modified, spectrally- discriminating fluorophores wherein the parent heterocyclic molecule is a dipyrrometheneboron difluoride compound.
  • Some BODIPY® fluorophores of the present invention have a BODIPY linker at the 3 position of the BODIPY® molecule that contains at least one functional group capable of attachment to AP-3 ribonucleotides, AP-3 deoxyribonucleotides or AP-3 dideoxyribonucleotides.
  • Specific BODIPY ® fluorophores useful in the present invention include BODIPY®s with adsorption maxima of about 450 to 700, and emission maxima of about 450 to 700.
  • Preferred embodiments include BODIPY®s with adsorption maxima of about 480 to 650, and emission maxima of about 480 to 650.
  • BODIPY®s examples include BODIPY® 503/512- SE (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid), BODIPY® 523/547 (4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s- indacene-3-propionic acid), BODIPY® 530/550 (4,4-difluoro-5,7-diphenyl-4- bora-3a,4a-diaza-s-indacene-3-propionic acid), BODIPY ® 558/568 (4,4- difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid), BODIPY® 564/570 (4,4-difluoro-5-styryl-4-bora-3
  • automated DNA sequencing refers to determining the sequence of a DNA strand of interest using an apparatus comprising an area having an electrophoresis gel, means for introducing labeled DNA fragments to the gel area, and photometric means for monitoring said labeled DNA fragments as they move through the gel.
  • Automated DNA sequencer refers to the instrument which is able to perform automated DNA sequencing.
  • sequencing primer means a synthetic oligonucleotide, restriction fragment, enzymatically-synthesized DNA fragment, or the like which hybridizes specifically to a region proximate to the region of DNA to be sequenced.
  • Universal sequencing primer refers to commonly-used primers known in the art, generally one that hybridizes specifically to a region of the M13 vector near the 5' end of the cloned insert. Specific examples of universal sequencing primers known in the art are -21M13, M13-40 and -36M13.
  • 5 1 position refers to the 5' position on the deoxyribose moiety of a poiynucleotide.
  • 3 1 position refers to the 3' position on the deoxyribose moiety of a nucleotide.
  • base attachment or “dye-terminator” refers to a molecule, particularly a fluorescent dye, attached to the C 7 position of a purine terminating base or the C 5 of a pyrimidine terminating base.
  • AP-3 or "AP-3 nucleotide” refers to the 3-amino-l- propynyl linker attached to the 5 position of pyrimidines or the 7 position of 7-deazapurines. See Figure 7.
  • BODIPY® linker or “BODIPY® functional group” refers to a substituted or unsubstituted alkyl containing one to thirty carbons and at least one functional group. Two different BODIPY® linkers are illustrated in Figure 1.
  • FAM shall refer to 5-carboxy-fluorescein
  • JOE shall refer to 2',7'-dimethoxy-4',5'-dichloro-6-carboxy-fluorescein
  • TAMRA shall refer to N,N,N',N'-tetramethyl-6-carboxy-rhodamine
  • ROX shall refer to 6-carboxy-X-rhodamine.
  • electrophoresis “lanes” or “tracks” or “columns” refers to the particular path in the electrophoretic medium in which the sequencing products are run.
  • the sequencing products terminating in dideoxyadenosine, dideoxycytodine, dideoxyguanosine or dideoxythymidine may be run in four separate lanes, or, if labeled differentially, in the same lane.
  • linkers or “linker arms” refers to molecules that tether a dye to a primer. Typical linker molecules include alkanes of various lengths.
  • automated GeneScanner refers to an instrument capable of performing analysis of fluorescently-labeled DNA or RNA.
  • Taqman or “Taqman assay” refers to assays that utilize the 5' - 3' exonuclease activity of Taq polymerase in a polymerase chain reaction to generate a specific detectable signal concomitantly with amplification.
  • An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5* end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay.
  • Annealing of the probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity.
  • the 5' - 3' exonuclease activity of Taq polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe.
  • the assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.
  • the oligonucleotide that is degraded has at least two light-emitting fluorophores attached. The fluorophores interact each other to modify (quench) the light emission of the fluorophores. The ⁇ '-most fluorophore is the quencher fluorophore.
  • the 3'-most fluorophore is the quenched fluorophore.
  • an oligonucleotide probe is labeled with a light-emitting quenched fluorophore wherein the reaction is carried out in the presence of a DNA binding compound (quenching agent) that interacts with the fluorophore to modify the light emission of the label.
  • quenching agent a DNA binding compound that interacts with the fluorophore to modify the light emission of the label.
  • labeled oligonucleotide refers to the oligonucleotide in the Taqman assay that is labeled with at least two BODIPY® fluorophores.
  • quenched refers to the interaction of the at least two BODIPY® fluorophores on the labeled oligonucleotide wherein when both BODIPY® fluorophores are present on the labeled oligonucleotide, fluorescence of either fluorophore is not detected.
  • quencher fluorophore refers to the BODIPY ® fluorophore at a position most 5' on the labeled oligonucleotide.
  • quenched fluorophore refers to the BODIPY ® fluorophore at a position most 3' on the labeled oligonucleotide.
  • quencher agent refers to intercalating compounds and the like similar to ethydium bromide for use in a Taqman assay similar to that used in the method of Fisher, et al., U.S. Pat. No. 5,491,063.
  • One novel aspect of the present invention is to provide a method for distinguishing polynucleotides having different 3 1 -terminal dideoxynucleotides in any method of DNA sequencing requiring electrophoresis of the products of the sequencing reactions, the method comprising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each poiynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled at the 5' position with a first fluorophore; each poiynucleotide in the second class having a 3'-terminal dideoxycytidine and being labeled at the 5' position with a second fluorophore; each poiynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled at the 5' position with a third fluorophore; and each poiy
  • Another aspect of the present invention allows BODIPY® fluorophores to be used in combination with prior art fluorophores and commercially-available software.
  • This method involves distinguishing polynucleotides having different 3'-terminal dideoxynucleotides in the chain termination method of DNA sequencing, the method comprising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each poiynucleotide in the first class having a 3'- terminal dideoxyadenosine and being labeled at the 5' position with
  • each poiynucleotide in the second class having a 3'-terminal dideoxycytidine and being labeled at the 5' position with BODIPY® 503/512 or FAM; each poiynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled at the 5* position with BODIPY® 558/568, BODIPY® 564/570 or TAMRA; and each poiynucleotide in the fourth class having a 3'-terminal dideoxythymidine and being labeled at the 5' position with BODIPY ® 581/591, BODIPY® 589/616 or ROX; wherein at least one of the classes is labeled with a BODIPY® fluorophore; electrophoretically separating on a gel by size the polynucleotides; illumina
  • a method of distinguishing polynucleotides having different 3'-terminal dideoxynucleotides in the chain termination method of DNA sequencing comprising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each poiynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled at the 5 1 position with a first BODIPY® fluorophore; each poiynucleotide in the second class having a 3'-terminal dideoxycytidine and being labeled at the
  • each poiynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled at the 5' position with a third BODIPY® fluorophore
  • each poiynucleotide in the fourth class having a 3'-terminal dideoxythymidine and being labeled at the 5' position with a fourth BODIPY® fluorophore
  • said first, second, third and fourth BODIPY® fluorophores are all different; electrophoretically separating on a gel by size the polynucleotides; illuminating with an illumination beam bands of said gel, said illumination beam being capable of causing said BODIPY ® fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the dyes.
  • said BODIPY® fluorophores have an adsorption maxima of about 450 to 700, and an emission maxima of about 450 to 700. In a more preferred embodiment, said BODIPY® fluorophores have adsorption maxima of about 500 to 625, and an emission maxima of about 500 to 625.
  • said 3'-terminal dideoxyadenosine is labeled at the 5' position with BODIPY® 523/547; said 3'-terminal dideoxycytidine is labeled at the 5' position with BODIPY® 503/512; said 3'-terminal dideoxyguanosine is labeled at the 5' position with BODIPY® 564/570; and said 3'-terminal dideoxythymidine is labeled at the 5' position with BODIPY® 581/591. Labeling the polynucleotides in this manner allows for the use of conventional, commercially-available software. However, it should be clear that one skilled in the art of computer software design that software could be altered such that the software could read different BODIPY® dyes attached to different classes of polynucleotides by way of different linker arm chemistries.
  • said chain termination method of DNA sequencing is performed by an automated DNA sequencing instrument.
  • the method of the present invention further includes the step of extending from a primer a plurality of polynucleotides by means of a DNA polymerase suitable for DNA sequencing or a reverse transcriptase suitable for DNA sequencing in the presence of dideoxyadenosine triphosphate, dideoxycytosine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate to form said first, second, third, and fourth classes of polynucleotides.
  • said DNA polymerase is selected from the group of ThermoSequenase, Klenow fragment, Sequenase®, Bst DNA polymerase, AmpliTaq® DNA polymerase, P i (exo-)DNA polymerase, rTth DNA polymerase or Vent(exo-)®DNA polymerase, and said reverse transcriptase is selected from the group of
  • RNA polymerase is used.
  • said BODIPY ® fluorophores are coupled to a primer suitable for sequencing by linkers.
  • said linker arms are selected from the group of (CH 2 ) 3 (CH 2 ) ⁇ , and (CH 2 ) 12 .
  • said poiynucleotide is labeled with more than one fluorophore, wherein said fluorophores include at least one BODIPY® fluorophore and at least one additional fluorophore.
  • said additional fluorophore has an adsorption maxima of about
  • said additional fluorophore is a BODIPY® fluorophore or FAM.
  • BODIPY® fluorophores for DNA sequencing wherein the BODIPY® fluorophore is attached at the 5' end of the products of the sequencing reaction and at the 3' end of the product of the sequencing reaction or at one or more internal positions of the products of the sequencing reaction.
  • a method for distinguishing poiynucleotide sequences in a hybridization method of DNA sequencing comprising the steps of: synthesizing a first, a second, a third and a fourth class of oligonucleotides, wherein all of said classes of oligonucleotides have a same length, said first, second, third and fourth classes of oligonucleotides differ from the oligonucleotides of each other class by one nucleotide base at a 3', a 5' or an internal position, and each oligonucleotide of the first class has a deoxyadenosine at said position and is labeled at the 5' position with a first fluorophore; each oligonucleotide in the second class has a deoxycytidine at said position and is labeled at the 5' position with a second fluorophore; each oligonucleotide in the
  • a method for genetic analysis of DNA fragments wherein said DNA fragments are labelled with at least one BODIPY® fluorophore.
  • Another important and novel aspect of the present invention is to provide a method for distinguishing polynucleotides having different 3'- terminal dideoxyribonucleotides in any method of chain termination DNA sequencing, the method comprising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each poiynucleotide in the first class having a 3'-terminal dideoxyadenosine triphosphate, said 3'-terminal dideoxyadenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-l-propynyl linker, said linker then attached to a BODIPY® linker at a 3 position of a first BODIPY® fluorophore that contains
  • Yet another embodiment of the present invention provides for the method of distinguishing polynucleotides having different ribonucleotides in any method of labelling polynucleotides by enzymatic incorporation, the method comprising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each poiynucleotide in the first class having an adenosine triphosphate, said adenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-l-propynyl linker, said Unker then attached to a BODIPY® linker at a 3 position of a first BODIPY® fluorophore that contains at least one reactive functional group; each poiynucleotide in the second class having a cytidine triphosphate, said cytidine triphosphate being attached at the 5 position ofthe pyrimidine to a 3-
  • a method for distinguishing polynucleotides having different deoxyribonucleotides in any method of labelling polynucleotides by enzymatic incorporation comprising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each poiynucleotide in the first class having a deoxyadenosine triphosphate, said deoxyadenosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-l-propynyl linker, said linker then attached to a BODIPY® linker at a 3 position of a first BODIPY® fluorophore that contains at least one reactive functional group; each poiynucleotide in the second class having a deoxycytidine triphosphate, said deoxycytidine triphosphate being attached at the 5 position of the
  • said adenosine triphosphate, dexoyadenosine triphosphate or 3 1 -terminal dideoxyadenosine triphosphate is labeled with BODIPY® 523/547 or BODIPY® 530/550; said cytidine triphosphate, deoxycytidine triphosphate or 3'-terminal dideoxycytidine triphosphate is labeled with BODIPY® 576/589, BODIPY ® 581/591, or BODIPY® 589/616; said guanosine triphosphate, deoxyguanosine triphosphate or 3" -terminal dideoxyguanosine triphosphate is labeled with BODIPY® 503/512; and said uracil triphosphate, deoxythymidine triphosphate or 3' -terminal dideoxythymidine triphosphate is labeled with BODIPY ® 558/568 or BODIPY® 564
  • Another aspect of the present invention allows BODIPY® fluorophores to be used in combination with prior art fluorophores and commercially-available software.
  • said internal labelling is performed by an automated GeneScanner.
  • Another important and novel aspect of the present invention is to provide an oligonucleotide substituted with at least two 4,4-difluoro-4- bora-3A,4A-diaza-s-indacene (BODIPY®) fluorophores for performing a
  • Taqman assay wherein a first 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY®) fluorophore is a quencher fluorophore and a second 4,4- difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY®) fluorophore is a quenched fluorophore.
  • BODIPY® 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene
  • a preferred embodiment of this aspect of the present invention provides BODIPY® 564/570 (4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s- indacene-3-propionic acid) as a quencher fluorophore.
  • An additional preferred embodiment of the present invention provides BODIPY® 576/589 (4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza- s-indacene-3-propionic acid) as a quencher fluorophore.
  • a further preferred embodiment of the present invention provides BODIPY® 581/591 (4,4-difluoro-5-(4-phenyl-l,3-butadienyl)-4-bora-3a,4a- diaza-s-indacene-3-propionic acid) as a quencher fluorophore.
  • Another preferred embodiment of the present invention provides BODIPY® 558/568 (4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-
  • Yet a further preferred embodiment of the present invention provides BODIPY® 581/591 (4,4-difluoro-5-(4-phenyl-l,3-butadienyl)-4- bora-3a,4a-diaza-s-indacene-3-propionic acid) as a quencher fluorophore.
  • An additional preferred embodiment of the present invention provides BODIPY® 503/512-SE (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-propionic acid) as a quenched fluorophore.
  • Yet another preferred embodiment ofthe present invention provides BODIPY® 523/547 (4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3- propionic acid) as a quenched fluorophore.
  • Another preferred embodiment of the present invention provides BODIPY® 530/550 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene- 3-propionic acid) as a quenched fluorophore.
  • Another aspect of the present invention provides an oligonucleotide substituted with at least one 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY®) fluorophores for performing a Taqman assay, wherein said at least one 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY ® ) fluorophore is a quenched fluorophore and a quencher agent is present in said Taqman assay. Any of the quenched (BODIPY®) fluorophores mentioned above can be used.
  • BODIPY® dyes can replace one or more prior art dyes.
  • DNA synthesis reagents were purchased from Applied Biosystems, Inc. (ABI) except ⁇ '-amino-modifier C3, C6, and C12 phosphoramidites were purchased from Glen Research.
  • Oligonucleotides R865, R932, R930, and R931 were synthesized trityl-on (0.2 ⁇ mole scale) using either an ABI model 380B or model 394 DNA synthesizer and purified using NensorbTM 20 columns according to the manufacturer's protocol (du Pont de Nemours & Co.).
  • NHS, TAMRA-NHS, and ROX-NHS ester were purchased from ABI.
  • 5- FAM-SE and BODIPY®-SE dyes were purchased from Molecular Probes and resuspended in anhydrous DMSO (50 mg/mL).
  • R930, R931, or R932 primers (0.6 ⁇ mole) were resuspended in 200 ⁇ L of 0.5 M NaHCO j /N ⁇ jCO a , pH 9.0 buffer and divided into seven aliquots.
  • RP-HPLC purification of oligonucleotides The RP-HPLC hardware system used consists of a Beckman model 127 gradient solvent module, a Rheodyne model 7125 injector, an Applied Biosystems (ABI) model 759A absorbance detector, and a Spectra-Physics model SP4600 DataJet integrator. Gradient RP-HPLC was performed using an ABI aquapore RP-300 column (4.6 mm x 250 mm) where "Buffer A” is 100 mM triethylammonium acetate (TEAA), pH 7.0, and "Buffer B” is 100 mM TEAA, 70% (v/v) acetonitrile.
  • Buffer A is 100 mM triethylammonium acetate (TEAA), pH 7.0
  • Buffer B is 100 mM TEAA, 70% (v/v) acetonitrile.
  • Dye-labeled oligonucleotides were purified using the following gradient conditions: 20% Buffer B, 5 min.; 20% - 40% Buffer B, 30 min.; 40% - 100% Buffer B, 18 min.; 100% Buffer B, 5 min. at a flow rate of 1.0 mL per min.
  • BODIPY ® fluorophores can substitute for conventional sequencing dyes or fluorophores.
  • the chemical structures of different fluorophores and their corresponding absorption/emission maxima are shown in Figure 1.
  • FAM, JOE, TAMRA, and ROX are four conventional fluorophores utilized in automated DNA sequencing.
  • substitution experiments were performed replacing conventional dye-labeled primers with
  • BODIPY®s that correspond to the emission spectrum ofthe prior art, dye- labeled primers.
  • Oligonucleotide R865, ( Figure 2) was dye-labeled with the fluorophores listed in Figure 1 and purified by RP-HPLC.
  • DNA sequencing reactions were generated by either solid-phase Bst sequencing or Taq cycle-sequencing. The results of the substitution experiment are shown in Figure 3.
  • three dye-labeled termination products i.e., FAM, TAMRA, and ROX
  • BODIPY® 589/616 reactions BODIPY * 503/512-, BODIPY * 523/547-, BODIPY * 530/550-, BODIPY * 558/568-, BODIPY * 564/570-, BODIPY * 576/589-, and BODIPY * 581/591-labeled termination products migrated approximately 3/4 to 1 base pair faster through the gel than FAM-, JOE-, TAMRA-, or ROX-labeled termination products, respectively.
  • the discrepancy between the two reactions is the result of the altered mobility of the different dye structures.
  • Oligonucleotides R930, R931, and R932 were dye-labeled with the BODIPY® dyes listed in Figure 1 and purified by RP-HPLC. As shown in Figure 3, increasing the linker arm length from (CH 2 ) ⁇ to (CH 2 ) 12 (R932) or addition of one 5' base plus (CH 2 ) 3 (R930) or (CH 2 ) ⁇ (R931) linker arm lengths slowed the mobility of BODIPY® 503/512-, BODIPY® 530/550-, and BODIPY® 564/570-labeled termination products.
  • BODIPY® 503/512-R930 labeled termination reactions mimicked the spacing pattern of FAM-R865, BODIPY® 523/547-R931 and BODIPY ® 530/550-R930 mimicked the spacing pattern of JOE-R865, BODIPY® 558/568-R930 and BODIPY® 564/570-R930 mimicked the spacing pattern of TAMRA-R865, and BODIPY® 576/589-R931, BODIPY® 581/591-R930, and BODIPY® 589/616-R865 mimicked the spacing pattern of ROX-R865, respectively, (compare highlighted boxes).
  • BODIPY® fluorophores alter the mobility of termination products in the same way, thus nullifying the need for chemical alteration of the fluorophore or software correction to generate accurate, evenly- spaced DNA sequences.
  • BODIPY® fluorophores leads to improved DNA sequencing in general and, due to their effect (or lack of differential effect) on electrophoretic mobility, the use of BODIPY® fluorophores leads to improved automated DNA sequencing in particular.
  • BODIPY"-SE dyes were resuspended in anhydrous DMSO (50 mg/mL), and BODIPY * 523/547-PA was converted to BODIPY * 523/547-SE according to the manufacturer's protocol.
  • dye-labeled primers were purified by reverse-phase high performance liquid chromatography (RP-HPLC). Fluorescent primers were resuspended in deionized (D.I.) water and diluted to 0.4 pmol/ ⁇ L.
  • the RP-HPLC hardware system used consists of a Beckman model 127 gradient solvent module, a
  • Dye-labeled oligonucleotides were purified using the following gradient conditions: 20% Buffer B, 5 min.; 20% - 40% Buffer B, 30 min.; 40% - 100% Buffer B, 18 min.; 100% Buffer B, 5 min. at a flow rate of 1.0 mL per min.
  • Figure 6 depicts the normalized emission spectra of four conventional dye-primers and BODIPY® dye-primers. It is important to note that all BODIPY® dyes were tethered to the primer via the tethers in Figure 2, and that no differential linker or nucleotide modification was required to achieve a precise, evenly-spaced, easily-read sequence reading.
  • doubly-labeled dye-primers were constructed and evaluated for fluoroescence energy transfer (ET).
  • ET fluoroescence energy transfer
  • oligonucleotides were systematically substituted with the acceptor dye at base increments away from either a FAM donor (0 to 3 bases apart) or a BODIPY * 503-512 donor (1 to 6 bases apart). It was observed that ET efficiency decreased with increasing distance, and decreased with decreasing spectral overlap between donor and acceptor dyes.
  • DNA synthesis reagents were purchased from ABI except ⁇ '-amino-modifier C3, C6, and C12, and amino modifier C6 dT phosphoramidites were purchased from Glen Research. Oligonucleotides FET and BET primers were synthesized trityl-on, using either an ABI model 380B or model 394 DNA synthesizer. BODIPY * 523/547 propionic acid (PA), and all BODIPY * -SE dyes were purchased from Molecular Probes.
  • PA propionic acid
  • BODIPY * -SE dye were resuspended in anhydrous DMSO (50 mg/mL), and BODIPY * 523/547-PA was converted to BODIPY * 523/547-SE according to the manufacturer's protocol.
  • the donor dye for the FET-3 primer ( ⁇ '-FAM-T ' GTAAAACGACGGCCAGT was synthesized (0.2 / /mole) using 6-FAM amidite and C6dT (T * ) and was ethanol precipitated.
  • the donor dye for the BET-3 primer ( ⁇ '-NTT ' GTAAAACGACGGCCAGT, was synthesized (0.2 ⁇ mole) using either C3 or C6 amino link (N) and C6dT (T * ) and resuspended in 200 ⁇ L of 0.1 N NaOH.
  • BODIPY * ⁇ 03/ ⁇ l2-SE was added, incubated at 2 ⁇ °C for 10 min., ethanol precipitated, incubated in 200 ⁇ L of 80% acetic acid for 20 min., and ethanol precipitated.
  • Both FET-3 and BET-3 primers were each resuspended in 160 ⁇ L of 0.2 ⁇ M NaHCO 3 /Na 2 CO 3 , pH 9.0 buffer and divided into four aliquots.
  • RP-HPLC The RP-HPLC hardware system consisted of a Beckman model 127 gradient solvent module, a Rheodyne model 712 ⁇ injector, an Applied Biosystems (ABI) model 7 ⁇ 9A absorbance detector, and a Spectra-Physics model SP4600 DataJet integrator. Gradient RP-HPLC was performed using an ABI aquapore RP-300 column (4.6 mm X 250 mm) where "Buffer A” is 100 mM triethylammonium acetate (TEAA), pH 7.0 and "Buffer B” is 100 mM TEAA, 70 % (v/v) acetonitrile.
  • Buffer A is 100 mM triethylammonium acetate (TEAA)
  • TEAA triethylammonium acetate
  • Buffer B 100 mM TEAA, 70 % (v/v) acetonitrile.
  • Dye-labeled oligonucleotides were purified using the following gradient conditions: 20% B, 5 min.; 20% B - 40% B, 30 min.; 40% B - 100% B, 18 min.; 100% B, ⁇ min. at a flow rate of 1.0 mL per min.
  • BET-3 dye- primers showed approximately the same signal strength compared to their single dye counterpart, but significant signal loss was observed for the FET-3 dye primers. Comparison of the normalized, overlapping spectral profiles of BET-3 dye-primers was indistinguishable from the single BODIPY * dye-primer spectra shown in Figure 3, consistent with efficient ET. Overall, the strong signal enhancement of the weaker fluorescent dyes contrasted with minimal enhancements of the normally stronger fluorescent dyes to produce a four dye-primer set with roughly balanced signal intensities.
  • the sensitivity of the complete BET-3 primer set was examined by serial dilutions of DNA template using an ABI 377A DNA sequencer on a single gel and sufficient signal was correctly analyzed even with a sixteen-fold reduction. This increased sensitivity of BET-3 dye-primers enables the direct loading of sequencing reactions onto gels without a previously-required laborious concentration step.
  • the unprocessed fluorescent signals generated from BET-3 sequencing reactions demonstrates the benefits of the uniform mobility, properly-balanced signal outputs and improved spectral purity of the present method.
  • the raw data from BET-3 reactions generates a DNA sequencing pattern that is visually interpretable and agrees well with the corresponding analyzed data. In contrast, no discernable sequence pattern could be detected from the unprocessed signals of conventional primers.
  • AP-3 nucleotides were purchased from DuPont NEN Products and dissolved and diluted to a final concentration of 10 mM. All
  • BODIPY ® -SE dyes were purchased from Molecular Probes and were resuspended in anhydrous DMSO (50 mg/mL).
  • RP-HPLC The RP-HPLC hardware system consisted of a
  • Dye- labeled ribonucleotides, deoxynucleotides or dideoxynucleotides were purified using the following gradient conditions: 0% B, 5 minutes; 0% B - 40% B, 30 minutes; 40% B - 100% B, 18 minutes; 100% B, ⁇ minutes at a flow rate of 1.0 mL per minute.
  • DNA synthesis reagents were purchased from ABI except ⁇ '-amino-modifier C3, C6, and C12 and amino modifier C6 dT phosphoramidites and 3'-amino-modifier CPG were purchased from Glen Research. Oligonucleotides BET primers were synthesized on 3'-amino- modifier CPG column trityl-on, auto-cleavage using either an BI model
  • BODIPY-SE dyes were purchased from Molecular Probes. BODIPY-SE dye were resuspended in anhydrous DMSO ( ⁇ O mg/mL).
  • B. Fluorescent primers The leader sequences for BET dye-primers are 5 * -NTGTT* or ⁇ '-NACGTTGT* followed by any primer sequence that is completely complementary to the target sequence. Primers were synthesized (0.2 ⁇ mole) using either C3 or C6 amino link (N) and C6dT (T*) and resuspended in 400 ⁇ l of 0.01 N NaOH.
  • BODIPY primers were resuspended in 200 ⁇ L of 0.2 ⁇ M NaHCO a /N ⁇ CO,, pH 9.0 buffer and 10 ⁇ L of either BODIPY ⁇ 88/ ⁇ 68-SE, BODIPY ⁇ 64/ ⁇ 70-SE, BODIPY ⁇ 76/ ⁇ 89-SE, BODIPY ⁇ 81/ ⁇ 91-SE, or BODIPY ⁇ 89/616-SE, was added and the mixtures were incubated at 25° C for 16 h. Following ethanol precipitation, dye-labeled primers were purified by RP-HPLC, resuspended in deionized (DI) water, and diluted to 0.4 pmol/ ⁇ l. C. RP-HPLC.
  • DI deionized
  • the RP-HPLC hardware system consisted of a Beckman model 127 gradient solvent module, a Rheodyne model 712 ⁇ injector, an Applied Biosystems (ABI) model 7 ⁇ 9A absorbance detector, and a Spectra- Physics model SP4600 DataJet integrator.
  • Gradient RP-HPLC was performed using an ABI aquapore RP-300 column (4.6 mm X 250 mm) where "Buffer A” is 100 mM triethylammonium acetate (TEAA), pH 7.0 and "Buffer B” is lOOmM TEAA 70% (v/v) acetonitrile.
  • Dye-labeled oligonucleotides were purified using the following gradient conditions: 20% B, 5 minutes; 20% B-40% B, 30 minutes; 40% B- 100% B, 18 minutes; 100% B, ⁇ minutes at a flow rate of 1.0 ml per minute.

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Abstract

L'invention concerne des procédés d'utilisation d'une catégorie de colorants, afin d'améliorer le séquençage d'ADN. On a décrit récemment une nouvelle catégorie de colorants, les fluorophores BODIPY®. La molécule hétérocyclique parente des fluorophores BODIPY® est un composé de difluorure de dipyrrométhènebore, qui est modifié afin de créer une catégorie importante de fluorophores à sélection spectrale. L'invention concerne des procédés d'utilisation d'ADN marqué au fluorophore BODIPY® servant à effectuer le séquençage d'amorces marquées au colorant, dans lequel les fluorophores BODIPY® sont fixés à l'extrémité 5' d'amorces de séquençage, des procédés de séquençage d'ADN utilisant de séquençage d'ADN en terminaison de chaîne, ainsi que de marquage interne de polynucléotides par incorporation enzymatique de ribonucléotides ou de désoxyribonucléotides à marquage fluorescent, ainsi que des oligonucléotides marqués par des composés de 4,4-difluoro-4-bora-3A,4A-diaza-s-indacène substitué (fluorophore BODIPY®), afin de mettre en application la méthode de Taqman. Les fluorophores BODIPY® présentent des caractéristiques spectrales améliorées par rapport aux colorants classiques de fluorescéine et de rhodamine. Les fluorophores BODIPY® possèdent une largeur de bande plus étroite, une insensibilité au solvant ou au pH, ainsi qu'une photostabilité améliorée et, de ce fait, permettent d'améliorer le séquençage et/ou la détection de l'ADN dans tout procédé nécessitant l'électrophorèse et la détection de l'ADN. De plus, les propriétés spectrales des fluorophores BODIPY® sont suffisamment semblables en longueur d'ondes et en intensité pour être utilisées avec l'équipement classique de l'état actuel de la technique.
EP96921749A 1995-06-23 1996-06-21 Nouvelles amorces marquees au colorant, ribonucleotides, desoxyribonucleotides et didesoxyribonucleotides servant a effectuer une analyse automatique d'adn et a mettre en application des methodes de detection et d'amplification homogenes Withdrawn EP0833936A4 (fr)

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
US08/494,216 US5614386A (en) 1995-06-23 1995-06-23 Alternative dye-labeled primers for automated DNA sequencing
US494216 1995-06-23
US540228 1995-10-06
US08/540,228 US5861287A (en) 1995-06-23 1995-10-06 Alternative dye-labeled primers for automated DNA sequencing
US553936 1995-11-06
US08/553,936 US5728529A (en) 1995-06-23 1995-11-06 Alternative dye-labeled ribonucleotides, deoxyribonucleotides, and dideoxyribonucleotides for automated DNA analysis
US612036 1996-03-07
US08/612,036 US5994063A (en) 1995-06-23 1996-03-07 Substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene compounds for homogenous amplification/detection assays
PCT/US1996/010729 WO1997000967A1 (fr) 1995-06-23 1996-06-21 Nouvelles amorces marquees au colorant, ribonucleotides, desoxyribonucleotides et didesoxyribonucleotides servant a effectuer une analyse automatique d'adn et a mettre en application des methodes de detection et d'amplification homogenes

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AU699939B2 (en) 1998-12-17

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