CA2225531A1 - Alternative dye-labeled primers, ribonucleotides, deoxyribonucleotides, and dideoxyribonucleotides for automated dna analysis and homogeneous amplification/detection assays - Google Patents

Abstract

Methods for the use of a class of dyes for improved DNA sequencing are provided. A new class of dyes, BODIPY fluorophores, has been described recently. The parent heterocyclic molecule of the BODIPY fluorophores is a dipyrrometheneboron difluoride compound which is modified to create a broad class of spectrally-discriminating fluorophores. The present invention provides methods for the use of BODIPY fluorophore-labeled DNA for dye-primer sequencing in which the BODIPYs are attached to the 5' end of sequencing primers, methods for DNA sequencing by the chain termination method of DNA sequencing and for internal labelling of polynucleotides by enzymatic incorporation of fluorescently-labeled ribonucleotides or deoxyribonucleotides, and provides oligonucleotides labelled with substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPYfluorophore) compounds for perfoming the Taqman assay. BODIPY fluorophores have improved spectral characteristics compared to conventional fluorescein and rhodamine dyes. BODIPY fluorophores have narrower band width, insensitivity to solvent or pH, and improved photostability, thus, BODIPY fluorophores lead to improved DNA sequencing and/or detection in any method where electrophoresis and detection of DNA is required. Additionally, the spectral properties of the BODIPY fluorophores are sufficiently similar in wavelength and intensity to be used with conventional equipment known in the art.

Description

WO 97/OOg67 PCII/US96/10729 ALTERNATIVE DyE~.ARF'.T.l;'.n Pl~ , RIBONUCLEOTIDES, DEOXYI~rRONlJCLEOTII)ES, AND DII)EOXYR~ONUCL13OTII)ES
FOR AUTO~IATED DNA ANALYSIS AND HOMOGENEOUS
A~PLIE'ICATION/DETECTION ~ AYS

This invent,ion was supported in part by a grant from the United States Gov~ le~t through the National In~iLu~es of Health (Grant Nos.
P30HG00210 (NIH) and T32HG00003 (NIH-NCHGR)). The U.S.
Govel~ ent has c ertain rights in this invention.

10~ l~LD OF T~F. INVENTION

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'D
fluorophore) compounds for hll~foved DNA sequencing by che~nic~l cleavage and by lhybridization, labelling of DNA fragments for genetic 1~analysis, improved DNA sequencing by the chain termination method of DNA sequencing and for internal labelling of polynucleotides by enzyl~latic incorporation of fluorescently-labeled ribonucleotides or deoxyribonucleotides, and performing the Taqman assay.
.

CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 KGROUND

The ability to determine the sequence of DNA is critical for underst~n~ling the function and control of genes and for applying many of the basic techniques of molecular biology. 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 ~ntip~rallel orientation by hydrogen bonds between complemeIl~sry bases of the nucleotides of the two strands:
deoxyadenosine triphosphate (A) pairs with thymidine triphosphate (T) and deo2~yguanosine triphosphate (G) pairs with dec,~y~ Lidine triphosphate (C).
The development of reliable methods for sequence analysis of DNA
(deoxyribonucleic acid) and RNA (ribonucleic acid) has been essential to the success of recomhin~nt. DNA and genetic engineering. When used 1~ with the other techniques of modern molecular biology, nucleic acid sequencing allows dissection of ~nim~l, plant and viral genomes into discrete genes with defined chemical structure. Since the function of a biological molecule is determined by its structure7 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 posscsscd by the original gene products.
The development of modern nucleic acid sequencing methods 2~; involved parallel developments in a variety of techniques. One was the emergence of simple and reliable methods for cloning small to medium-sized strands of DNA into bacterial plasmids, bacteriophages, and small ~nim~l viruses. Cloning allowed the production of pure DNA in sufficient quantities to allow chemical analysis. Another was the use of gel electrophoretic methods for high resolution separation of oligonucleotides WO 97/OOg67 PCT/US96/10729 ~ on the basis of size. The key development, how~v~r, was the introduction of methods of generating ~ets of fr~men~s of cloned, purified DNA that contain, in their collection of lengths, the information necçss~ry to define the sequence of the nucleotides comprising the parent DNA molecules.
Presently there are several approaches to DNA sequence determin~ion, see, e.g, the dideoxy chain termin~tion method, Sanger et al., Proc. Natl. Acad. Sci., 74:5463-67 (1977); the cheInic~l degradation method, M~Y~m et al., Proc. Natl. A~ad. Sci., 74:560-564 (1977); and hybrirli~t.ion methods, Drmanac et al., Genomics, 4:114-28 (1989), Khrapko, FEB 266:118-22 (1989). The chain termin~t.ion method has been i~ ov~d in several ways, and serves as the basis for all currently available automa~ed DNA seqlle~cing m~-!hine~. ,See, e.g, Sanger et al., J. Mol. Biol., 143:161-78 (1980); Schreier et al., J. Mol. Biol., 129:169-72 (1979); Smith et al., Nucleic Acids Research, 13:2399-2412 (1985); Smith 1~; et al., Nature, 321:674-79 (1987) and U.S. Patent No. 5,171,534; Prober et al., Science, 238:336-41 (1987); Section II, Meth. Enzymol., 155:51-334 (1987); Church et al., Science, 240:185-88 (1988); Swerdlow and Gesteland, Nucleic Acids Research, 18: 1415-19 (1989); Ruiz-Martinez et al., Anal.
Chem., 2851-58 (1993); Studier, PNAS, 86:6917-21 (1989); Kiele~ wa et al., Science, 258::L787-91; and Connell et al., Biotechniques, 5:342-348 (1987).
The method developed by Sanger is referred to as the dideoxy chain termin~t.ion method. In a commonly-used variation of this method, a DNA
seFment i8 cloned into a single-stranded DNA phage such as M13. 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 fragrnent isolated from the parental recombinant DNA that hybridizes specifically to a region of ~ the M13 vector near the 3' end of the cloned insert. In each of four sequencing reactions, the primed synt.he~i~ is carried out in the presence of enough of the dideoxy analog of one of the four possible CA 0222~31 1997-12-22 WO 97/OOg67 ~ PCT/US96/10729 deo~ynucleotides so that the growing chains are randomly termin~t,ed by the incorporation of these "~ie~(len~ nucleotides. The relative concentration of dideoxy to deo~y forms i8 adjusted to give a spread of termination events COr~ e~onding to all the possible chain lengths that can ~; be resolved by gel electrophoresis. The products from each of the four primed synthe~ reactions are loaded into individual lanes and are separated by polyacrylamide gel electrophoresis. R~lio~ tive 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 P~min~tion of the pattern of bands in the four lanes. Because the products from each of the four syntllRsi~ reaction~ must be run on separate gel lanes, there are problems with comparing band mobilities in the different lanes.
1~ Turning to automated DNA sequencing m~chines, in general, fr~ me~s having different termin~ting bases can be labeled with different fluorescent dyes, which are ~t,t~hed either to a primer for dye-primer seqlleI cing in which the fluol esc~lt dyes are ~tt~ched to the 6' end of the primers, e.g., Smith et al. (1987, cited above), or to the base of dideoxynucleotides for dye terminator sequencing in which the fluorescent dyes are attached to the C7 position of a purine termin~qting base and the C6 of a pyrimidine termin~ting base, e.g., Prober et al. (cited above). A
fluorescence tletector then can be used to detect the fluorophore-labeled DNA fr~gment~. The four different dideoxy-termin~ted 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 m~im~ Each of these tags is coupled chemically to the primer used tG
initiate the synthesis of the 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 =

WO 91/00~67 - PCT/US96/10729 ~ fr~-n.snt~ are then combined and loaded onto the same gel column for electrophoretic se~al dlion. Base sequence is determined by analyzing the fluorescent ~ emitted by the fr~Fnent.~ as they pass a stationary de~ ~or during the separation process.
~; Obt~ining a set of dyes to label the different fragments i8 a majordifficulty in automated DNA sequencing systems. First, it i8 dif~lcult to ~md three or more dyes that do not have emission bands which overlap ~ nific~ntly since the typical emi~ion band halfwidth for organic fluol~ecellt dyes i3 about 40-80 nanometers (nm) and the width of the visible spectrum is only about 350-400 nm. Second, even if dyes with non-overlapping emi~R;on bands are found, the set may still be unsuitable for DNA ~eqll~ncin~ the respective fluorescent efficiencies are too low. For ~Y~mple, Pringle et al., DNA Core F~cilities Newsletter, 1:15-21 (1988), present data in(li~s~tinF that increa~ed gel lo~(1in~ cannot compe~te low 1~ fluolesce~lt emci~r-cies.
Another difit-lculty with obt~inin~ an appropriate set of dyes is that when several fluorescent dyes are used concurrently, f?~cit~t.ion becomes difficult because lthe absorption bands of the dyes are often widely separated. The most efficient excitation occurs when each dye i~
illllmin~ted at the wavelength corresponding to its absorption band m~imum. Thus, one often is forced to compromise between the sen~il,ivi~y of the detection system and the increased cost of providing separate excitation sources for each dye. In ~ itiQn, when the number of differently sized fragmenLts 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 conform~tio~ of the dyes and linkers must not effect adver~ely the electrophoretic mobilities of closely-sized fragments. Changes in electrophoretic mobility can result in extensive $0 band broadening or ~ ~vel sal of band positions on the gel, thereby destroying the correspondence between the order of bands and the order CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 of the bases in the nllclai~ acid sequence. Due to the many problems associated with altered electrophoretic mobility, correction of mobility discrep~ncies by computer software is necessaly in prior art systems.
Finally, the fluolcscellt dyes must be comr~qtihle with the chemi~try used 5s to create or manipulate the fragments. For example, in the chain termination method the dyes used to label primers and/or the dideoxy chain terminators must not interfere with the activity of the polymerase or reverse transcriptase employed.
Because of these severe constraints, only a few sets of fluorescent dyes have been found that can be used in DNA sequencing, particularly autom~ted DNA sequencing, and in other diagnostic and analytical techniques, e.g., Smith et al. (1985, cited above); Prober et al. (cited above); Hood et al., European patent application 8500960; Bergot et al.
(cited above); Fung et al. (cited above); Connell et al. (cited above); Lsee, et al., NucleicAc~ds Research, 20:2471-83 (1992); and M~nl~h~n et al., U.S.
Patent No. 5,188,934.
In view of the above, DNA sequencing would be advanced significantly by the availability of new sets of fluorescent dyes which (1) are physiochemically similar, (2) permit ~tecti~n 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 ~mer~hle 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).
Until the present inventionS one problem encountered was that each fluorophore altered the "normal" electrophoretic mobility of the corresponding termination products during gel electrophoresis such that software correction files were needed to generate accurate, evenly-spaced DNA sequences. See, Smith et al., Nature, 321:674-79 (1986) and U.S.
Patent No. 5,171,534. Thus, the set of discrimin~ting fluorophores CA 0222~31 1997-12-22 described in the literature is small, snd the search for i~ o~ed, alternative dyes has been difficult at best.
There are several different chemical mor~ific~t.ions that have been ~I,L~ .ted to correct for differences in gel mobility between different dye-labeled primer~ in automated DNA seqll~n~ing. Generally, fluorescein and its del-ivaliv~ dyes labeled in DNA seq~lencing reactions have different gel mol~ilitia~ in comparison to rho~l~mine and its derivative dyes labeled in DNA seq~lellcing reactions. Fluolesceill and its derivative dye-labeled reactions typically move through the gel faster (sometimes greater than one base position) than rho~l~mine and its del iv~i~ive dye-labeled reactions.
For ex~mple, if u~ing the -21M13 universal sequencing primer, 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 rho~l~mines are coupled to the primer using six-carbon amino linh;er arm. Mobility collec~ion software, ho~v~ver, is required ~ itinnally to generate properly spaced DNA termination fr~nent.~. Another aY~mpla involves custom sequencing primers. These primers refer to any oligonucleotide sequence that can act as a suitable DNA sequencing primer. To all custom sequencing primers, a 5'-leader sequence (5'-CAGGA) must be coupled to the primer and custom sequencing primer~ must use the M13RP1 mobility correction software to generate properly-spaced DNA ter~nin~t.inn fr~nent~. The leader sequence is the first five bases of the reverse M13RP1 seqlle~cing primer.
M13RP1 is the mobility software file used to generate properly spaced DNA termin~t.ion fr~grnentq for the reverse sequencing primer.
Aside from DNA sequencing, a signi~lcant advance in DNA
manipulation was tlhe development of the polymerase chain reaction (PCR) technique as disclosed in U.S. Pat. Nos. 4,683,196; 4,683,195; and 4,800,1~9. The term "polymerase chain reaction" or "PCR" refers generally to the procedure involving: (1) treating extracted DNA to form single-stranded complama~ ry strands; (2) adding a pair of oligonucleotide CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 primers, wherein one primer of the pair i8 sub~t~ntis~11y complemant~ry to part of the sequence in the sense strand and the other primer of each pair is subst~nti~11y complementary to a different part of the same sequence in the compleme~t~ry ~ntiRen~e strand; (3) ~nne~1inF the paired primers to the compleme~tDry sequence; (4) simultaneously extending the slnne~1e~1 primers from a 3' terminus of each primer to synt.hefii7e an extension product compleme~t~ry to the strands ~nn~1etl to each primer wherein said ~t~n~ n products after separation from the complement serve as te~nrl~tes for the synth~iR of an extension product for the other primer of each pair; (5) sepalali~lg said ~Y~en~ion products from said templates to produce single-stranded molecules; and (6) amplifying said single-stranded molecules by repeating at least once said ~nne~1in~, eYteIl~linF and separating steps.
Detection methods generally employed in st~n-l~rd PCR techniques use a labeled probe with the ~mr1ified DNA in a hybridization assay.
Other assays include the use of fragment length polymorphism (PCR
FLP), hybri(li7~tion to allele-specific oligonucleotide (ASO) probes (Saiki et al., Nature 324:163 (1986)), or direct sequencing via the dideoxy method (using amplified DNA rather than cloned DNA). The st~n~rd PCR
technique operates by rep1ic~t.ing a DNA sequence positioned between two primers, providing as the major product of the reaction a DNA sequence of discrete length termin~ting with the primer at the 5' end of each strand. Thus, insertions and deletions between the primers result in product sequence of different lengths, which can be detected by sizing the 26 product in PCR-FLP. In an ~Y~mple of ASO hybridization, the amplified DNA is fixed to a nylon filter (by, for example W irradiation) in a series of "dot blots", then allowed to hybridize with an oligonucleotide probe labeled with HRP under stringent conditions. After w~hing, tetrametholbenzidine (TMB) and hydrogen peroxide are added- HRP
oxidizes the hydrogen peroxide, which in turn oxidizes the TMB to a blue precipitate, in-lir~t.inF hybridized probe.

CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 While the PCR technique as presently practiced is an ~ el~ely pow~rrul method ror amplifying nucleic acid sequences, the detection of the amplified material requires additional manipulation and subsequent h~ncllin~ of the PCR products to determine whether the target DNA is present. It i8 desirable to decrease the number of subsequent handling steps required ~;~rle~ly for the detection of ~mplified material.
Holland, et al., PNAS 88:7276-7280 (1991) de~cribe an assay known as a Taqman assay. The 5' - 3' e2~0nllcle~e activity of Taq polymerase i8 employed in a IDolymerase chain reaction product detection system to lLO generate a specific detectable signal concomi~ntly with amplification. An oligonucleotide probe, non~Ytenc~hle at the 3' end, labeled at the 5' end, and ~lesi~ned to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. ~nn~lin~ of the probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity.
During amplific~qt.iQn, the 6' - 3' exonuclease activity of Taq polymerase degrades the probe into smaller fr~gmeIlt~ that can be differenti~terl from n~eFraded probe. The assay is sensitive and specific and is a significant hlll3l 0v~ment 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. Patent No. 5,210,015 to Gelfand, et al., and Holland, et al., PNAS
88:7276-7280 (1991) are hereby incorporated by reference.
Further, 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 Z5 results in the clea~vage 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 CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 -1~
particular, to homogeneous ~mplification/detection assays where hybridized probe is cleaved concomitant with primer extension. A
homogeneous ~mplific~tion/detection assay is provided which allows the simultaneous ~letection of the accumulation of amplified target and the sequence-specific detection of the target sequence. U.S. Pat. No.
~,491,063 to Fisher, et al. is hereby incorporated by reference. Further, Lee, et al., NAR 21:3761-3766 (1993) describe nick tr~n~l~t.ion PCR using fluorogenic probes. In this assay, two probes were used to detect mutant and wildtype cystic fibrosis alleles. Lee, et al., NAR 21:3761-3766 (1993) is incorporated herein by reference.
A new class of dyes, BODIPY~ fluorophores, has been recently described. See, ~llFl~ntl~ et al., Moleculor Probes: Nandbook of Fluo~sce~t 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 dipyrl ollletheneboron difluoride compound and which is modified to create a broad class of spectrally-discrimin~t.in~ fluorophores, see Figure 1. The conventional n~ming of these dyes is BODIPY~
followed by their a~ .xi ~ t,e absorption/emission m~im~) e.g, BODIPY~\
530/B~0. The present invention provides for BODIPY2 fluorophores for methods for DNA seqllencing by chernic~l cleavage, hybri~ tioll, chain termination, for genetic analysis and for performing the Taqman assay.
In addition to the specifically-cited references above, additional prior art techniques include the following:
U.S. Patent No. 4,318,846 to Kh~nn~ 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.

CA 0222~31 1997-12-22 WO 97/00~67 PCI/US96/10729 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 Furlg 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 rho~mines, 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 rho.l~mines with different substitutions, and separated by gel electrophore~is.
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 1~ differentially-labeled with fluorophores, all fluoresceins with different sul~ ulions, 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 fr~Fments produced in DNA sequencing operations. The system comprises a source of chromophore or fluorescent tagged DNA fr~Frnent~, 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 fr~meIlts as they move through the gel.
U.S. Patent No. 5,366,603 is drawn to automatic DNA sequencing 7,5 wherein the DNA is marked with near infrared fluorescent d~es.
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 ~tt~ ed to the BASE entity at a position other than the C5 position when BASE is a pyrimidine, at a position other than the c8 CA 0222~31 1997-12-22 position when BASE is a purine and at a position other than the C7 position when BASE is a 7-deazapurine.
U.S. Patent No. 4,755,458 to Rabbani, et al., is drawn to compositions for ~Ptectin~ the presence of a nucleotides sequence of ~; interest. The composition includes a first polynucleotide molecule is sub~t~nti~1ly comrl~meIlt~ry to and capable of hybridizing with a specific sequence of interest and which is labeled with a first detectable marker;
a second polynucleotide molecule is not subst~n1;iiqlly 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 polynucleotide molecule that is sllh~tont.i~lly compl~mellt~ry to or subst~ntj~lly i(l~nt.ic~l to the second polynucleotide but is unlabeled or labeled with a second detectable marker.
U.S. Patent No. 5,151,507 to Hobbs, et al., drawn to alkynylamino-1~; nucleotides useful as chain termin~.in~ substrates for DNA sequencing.
U.S. Patent No. 5,274,113 to Kang, et al., is drawn to deliv&~ives of di~yl-lonletheneboron difluoride fluorescent dyes that can be ~tt~l.hed to nucleic acids, proteins, carbohydLates and other biologically-derived materials. The compounds of Kang, et al., show various functional groups for ~tt~.hment of the di~yl-lol~letheneboron difluoride fluorescent dyes to the biologically-derived materials.

SIJMMARY OF THE INVENTION

BODIPY8' fluorophores have improved spectral characteristics compared to conventional fluorescein and rhodamine dyes. The BODIPY~' fluorophores have narrower band width, insen:~ilivi~y to solvent or pH, and improved photostability. Thus, the use of BODIPY'!9 fluorophores leads to improved DNA sequencing or analysis of DNA fra~ ents in any method where electrophoresis of BODIPY~-labeled DNA is required.

-CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 It i8 an object of the present invention to provide methods for the use of a class of dyes particularly suited for DNA sequencing.
It is an ~iti~nal particular object of the present invention to provide methods for the use of BODIPY0 fluorophores in the chemic~l ~; cleavage method o~ DNA sequencing.
It is a particlllar object of the pre~ent invention to provide methods for the use of BOD][PY0 fluorophores for any method of DNA sequencing in which polynucleotide products of the seqll~ncin~ 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~D fluorophores which have been chemi~lly-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. BODIPY0 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.
If BODIPY~ fluorophores are used in four out of the 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 BODIPYE~ fluorophores alter the mobility of the corl e~onding termlin~tion products in the same way, nullifies the need for software correction to generate evenly-spaced DNA sequences.
An ~ itional object of the present invention is to provide methods for the use of BODIPY0 fluorophores for DNA sequencing wherein the 2~; BODIPY'9 fluorophore i8 ~t~~h~d at the 5' end of the polynucleotide product of the sequencing reaction and at the 3' end or at one or more internal positions of the products of the sequencing reaction.
Thus, in accompli~hin~ the foregoing objects, there is provided a method for analysis of DNA fragments wherein said DNA fragments are labelled with at least one BODIPYX fluorophore. Further, in accompli~hing the ~oregoing objects, there is provided in accordance with CA 0222~31 1997-12-22 the present invention, a method for disting~ hin{~ polynucleotides having different 3'-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 polynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled with a first fluorophore; each polynucleotide in the second class having a 3'-terminal dideu~y~idine and being labeled with a second fluorophore; each polynucleotide in the third class having a 3'-terminal dideoxygll~n- sine and being labeled with a third fluorophore; and each polynucleotide in the fourth class having a 3'-terminal dideo~ylhymidine and being labeled with a fourth fluorophore; wherein at least one of said fluorophores is a BODIPY~I\ fluorophore, and, wherein if said first, second, third and fourth fluorophores are all different, said polynucleotides can be electrophoresed in a same or different lanes; or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes; electrophoretically sel~a~ g on a gel by size the polynucleotides; illllmin~ting with an illllmin~t.ion beam the bands on the gel, the illllmin~t.ion beam being capable of c~ ing the fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.
It is another, particular object of the present invention to provide BODIPY0 fluorophores for DNA sequencing wherein the BODIPY~
2~; fluorophore is ~t+~chad to a nucleotide at a 3' BODIPYX position.
It is a further object of the present invention to provide methods for the use of a class of dyes particularly suited for the chain termination method of DNA sequencing. It is also an object of the present invention to provide methods for labelling internally RNA or DNA fragments by enzymatic incorporation of dye-labeled ribonucleotides or deoxynucleotides. The labeled fragments may then be analyzed.

WO 97/00~67 - PCT/US96/10729 -1~
Further in accompli~hing the foregoing objects, there i8 provided a method for analysis of DNA fr~E~ments wherein said DNA fr~gmeI ~s are labeled with at least one BODIPY~ fluorophore. In accompli~hing the foregoing objects, there is provided in accordance with the present invention, a method for distin~1i~hin~ polynucleotides having different 3'-terminal dideo2~yribonucleotides in any method of chain termin~t.ion DNA
seqller-cin~, the method COll,yl ising the steps of: forming a mixture of a ~lrst, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having a 3'-terminal dideoxy~tle~osin~
triphosphate, said 3'-terminal dideo2~yadenosine triphosphate being ~tD.rh~d at the 7 position of the 7-deazapurine to a 3-amino-1-~1o~y~lyl linker, said linker then ~t,t~ched to a BODIPY~ linker at a 3 position of a first BODIPY~I9 fluorophore that contains at least one reactive functional group; each polynucleotide in the second class having a 3'-terminal 1~ dideo~y.;y~idine triphosphate, said 3'-terminal didec.~y~idine triphosphate being ~tt~ched at the 5 position of the pyrimi~ine to a 3-amino-1-~-o~y~lyl linker, said linker then ~thched to a BODIPY~ linker at a 3 position of a second BODIPY~' fluorophore that contains at least one reactive functional group; each poly-nucleotide in the third class having a 3'-terminal dideoxygll~nosin~ triphosphate, said 3'-terminal dideoxyguanosine triphosphate being ~thched at the 7 position of the 7-~e~7.~purine to a $-amino-1-~o~y~lyl linker, said linker then ~tt~rhed to a BODIPY~' linker at a 3 position of a third BODIPY0 fluorophore that contains at least one reactive functional group; each polynucleotide in the 5~5 fourth class having a 3'-terminal dideoxythymidine triphosphate, said 3'-terminal dideoxythymidine triphosphate being ~tt~ch~d at the 5 position of the pyrimidine to a 3-amino-1-1Jl o~y~lyl linker, said linker then attached to a BODIPY~ linker at a 3 position of a fourth BODIPY'19 fluorophore - that contains at least one reactive functional group; wherein if said first, second, third and fourth BODIPY~ fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane; or CA 0222~31 1997-12-22 -1~
wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes; electrophoretically separating on a gel by size the polynucleotides; ill.lmin~t.ing ~,vith an ill~lmin~tion beam the bands on the gel, the illl]min~tion beam being capable of c~ in~ the fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.
Additionally, in accomrli~hing the foregoing object~ there is provided a method for disting~ hing 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 poly-nucleotides, each poly-nucleotide in the first class having an adenosine triphosphate, said adenosine triphosphate being ~ttD(~hed at the 7 position of the 7-tle~ u~h~e to a 3-amino-1-~lo~yl~yl linker, said linker then ~tt~che-l to a BODIPY~) linker at a 3 position of a first BODIPY~ fluorophore that contains at least one reactive functional group; each polynucleotide in the second class having a cytidine triphosphate, said cytidine triphosphate being ~tt~ched at the 5 position of the py-rimi(line to a 3-amino-1-~1 o~yl,yl linker~ said linker then ~tt~ched to a BODIPY~ID linker at a 3 position of a second BODIPY~ fluorophore that contains at least one reactive functional group; each polynucleotide in the third class having a guanosine triphosphate, said gll~nosine triphosphate being attached at the 7 position of the 7-tle~purine to a 3-amino-1-~io~y"yl linker, said linker then attached to a BODIPY~ linker at a 3 position of a third BODIPY'ID
fluorophore that contains at least one reactive functional group; each polynucleotide in the fourth class having a uracil, said uracil triphosphate being ~tt~ched at the ~; position of the pyrimidine to a 3-amino-1-p~ o~yllyl linker, said linker then ~tt~ched to a BODIPYal~ linker at a 3 position of a fourth BODIPY~ fluorophore that contains at least one reactive functional group; wherein if said first, second, third and fourth BODIPY'19 CA 0222~31 1997-12-22 fluorophores are all different, said polynucleotides can be electrophoresed in a same or a dirrel~.,t lane; or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;
electrophoretically separating on a gel by size the polynucleotides;
~ min~t.in~ with an illllmin~tion beam the bands on the gel, the illllmin~t.ion beam being capable of c~ in~ the fluorophores to fluoresce;
and identify-ing the classes of poly-nucleotides in the bands by the fluolescel.ce or absorption spectrum of the fluorophores.
Additionally, in accomplishing the foregoing objects, there is provided a method for a method for disting~ hing polynucleotides having different deo~yribonucleotides in any method of labelling poly-nucleotides by enzymatic incorporation, the method comprising the steps of: forming a mixture of a first, a second, a third, and a fourth class of poly-nucleotides, each poly-nucleotide in the first class having a deoy~e~osine tliphosphate, said deoyadenosine triphosphate being ~tt~ch~d at the 7 position of the 7-deazapurine to a 3-amino~ o~yllyl linker, said linker then ~t.t~qche~l to a BODIPY~ linker at a 3 position of a first BODIPY~' fluorophore that contains at least one reactive functional group; each poly-nucleotide in the second class having a deo~y~;y~idine triphosphate, said. deu~y~idine triphosphate being ~tt~chad at the 5 position of the py-r;mi~line to a 3-amino-l-~lo~yllyl linker, said linker then sltt~ha~l to a BODIPY0 linker at a 3 position of a second BODIPY0 fluorophore that contains at least one reactive functional group; each poly-nucleotide in the third class having a deoxyguanosine triphosphate, said deoxyguanosi:ne triphosphate being attached at the 7 position of the 7--le~ purine to a 3-amino-1-~1o~yllyl linker, said linker then attached to a BODIPY9' linker at a 3 position of a third BODIPY~ fluorophore that contains at least one reactive functional group; each poly~ucleotide in the fourth class having a deoxythymidine triphosphate, said deoxythymidine triphosphate bein~ ~tt~ched at the 5 position of the py-rimidine to a 3-CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 -1~
amino~ yllyl linker, said linker then ~tt~ched to a BODIPY~' linker at a 3 position of a fourth BODIPY~ fluorophore that contains at least one reactive functional group; wherein if said first, second, third and fourth BODIPY~ fluorophores are all different, said polynucleotides can be ~i electrophoresed in a same or a different lane; or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes; electrophoretically separating on a gel by size the polynucleotides; illllmin~t.ing with an illllmin~t.i~ n beam the bands on the gel, the illllmin~t.ion beam being capable of c~ in~ the fluorophores to fluoresce; and idelltiryillg the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.
It is an ~ litional object of the present invention to provide oligonucleotides labelled with substituted 4,4-difluoro-4-bora-3A,4~-diaza-s-indacene (BODIPY~ fluorophore) compounds for performingthe Taqman assay.
Other and further objects, features and advantages will be apparent and the invention more readily underfitood from a reading of the following specification and by reference to the accompanying drawings forming a 20 part thereof, wherein the eY~mrl~fi of the presently preferred embodiments of the invention are given for the purposes of disclosure.

DES~ llON OF THE DRA~1VINGS

Figure 1: Chemical structures of several DNA sequencing fluorophores are shown.

Figure 2: 5'-end mo.lific~t.ions of (A) single dye-labeled primers:
R865, R932, R930, and R931; and (B) 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-WO 97/OOg67 PCI~/US96/10729 -1~
503/512. Followingremoval of the monometho,~y~ yl group, BET primers were end-labeled with the BODIPY' dye set. The (CH2)n for BET primers COI-- e j~ond to (CH2)~ for BODIPY' 581/691 and (CH2)6 for BODIPY-503/512, BODIPY- 523/547, and BODIPY 564/570 dyes.

Figure 3: Depicts the results of a dye-labeled su~LiLulion experiment. DNA ~equencing reactions were generated by solid-phase Bst sequencing. The region shown corresponds to a~lo~ ely 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 l max (parenthesefi) for dye-primers was determined using a Model F-4010 fluorescence spectrophotometer (F~it~chi, Ltd) in lX TBE buffer (0.089 M Tris-borate, 0.002 M Na2EDTA) cont~inin~ 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 inten~ity values were determined by norm~li7ing the BODIPY dye signal to the rem~ining dye sign~l~ and comparing it to its norm~ e-l conventio~l dye signal. Spec.
measurements were performed in duplicate and determined by comparing the fluorescence inteI~ity at l max of BODIPY dye-primers to conventional dye-primers. FAM and BODIPY 603/512 were excited at 488 nm and all rem~ining dyes were at 514 nm.

2E; 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 BODIPY0 primers contain BODIPY~ 503/512-"C", BODIPY0 WO 97/OOg67 PCT/US96/10729 530/650-"A", BODIPY'19 664/570-"G", BODIPY~ 581/591-"T" dye labels.
Arrows above the sequence chromatograms highlight base calling errors, and the a~r..xi~ te base regions from the primer peak are listed.

Figure 5: A general synthetic scheme for end labeling (Route I) and ~; internal labeling (Route II) BODIPY~ phosphoramidites is depicted. For specific BODIPY'19 chemical structures, see Figure 1.

Figure 6: Depicts normalized emission spectra of four conventional dye-primers and BODIPY~ dye-primers.

Figure 7: Chemical structures of AP-3 nucleotides are shown, where Rl=OH and R2=OH for ribonucleotides; Rl=OH and R2=H for deoxynucleotides; and I2q=H and R2=H for dideoxyribonucleotides.

Figure 8: (A) Double dye-labeled primers. Since different protecting groups block the linker arm ~minefi, BODIPY energv transfer (BET) primers were first labeled internally with BODIPY ~03/512, BODIPY 523/547 or BODIPY 530/550. After removal of the monomethul~yL. ;~yl group, BET primers were end-labeled with the BODIPY dye set. For BODIPY 503/512, BODIPY 523/547 or BODIPY
530/550 dyes, n = 6, R1 = CH3, and R2 = (CH2)~NHBODIPY. For BODIPY 658/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, and BODIPY 589/616 dyes, n = 3, R1 = (CH2)GNHBODIPY and R2 = CH3.
Primers are blocked at the 3' end with a modified amino group (NH2) to prevent polymerase extension of the probe. (B) Diagnostic application in determining reverse transcriptase resistance to antiviral drug therapy in 2~S patients infected with human immunodeficiency virus type-1 (HIV-1).
Drug resistant markers have been previously described by B.A. Larder, "Reverse Transcriptase", A.M. Skalka and S.P. Gof$ Eds., pp. 20~-222 (Cûld Spring Harbor Labûratory Press, 1993). BET probe labeled with WO 97/00~67 PCT/US96tlO729 BODIPY 503/512 iLs specific for the wild-t~pe sequence and BET probe labeled with BODIPY 523/547 i8 specific for the drug resistance sequence.
..
The dlc-whlgs and ~lgures are not to scale and certain features mentioned may be exaggerated in scale or shown in schematic form in the interest of claril~y and conciseness.

nET,~J,F'.T~ DESClRIPTION OF THE INVENTION

It will be apparent to one skilled in the art that various su~s~i~ulions and mo(~ c~tions may be made to the invention disclosed herein without departing from the scope and the spirit of the invention.
:10 As used herein, "BODIPY~" shall refer to a class of modified, spectrally- discrimin~t.ing fluorophores wherein the parent heterocyclic molecule is a dil~yr~ etheneboron difluoride compound. Some BODIPY~
fluorophores of t~e present invention have a BODIPY linker at the 3 position of the BODIPY~ nnolecule that contains at least one functional group capable of ~tt7~chm~nt to AP-3 ribonucleotides, AP-3 deo~yribonucleoticles or AP-3 dideoxyribonucleotides. Specific BODIPY~
fluorophores useful in the present invention include BODIPY6's with adsorption m~im~ of about 450 to 700, and emission m~im~ of about 450 to 700. PreferTed embodiments include BODIPY~s with adsorption m~im~ of about 480 to 650, and emission m.q~im:V~ of about 480 to 650.
F.~mrles of prefeITed embodiment BODIPYDs include BODIPY~9503/512-SE (4,4-difluoro-6,7-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-.25 bora-3a,4a-diaza-s-indacene-3-propionic acid), BODIPY6' 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-3a,4a-diaza-s-indacene-3-propionicacid),BODIPY~'576/589 (4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-CA 0222~31 1997-12-22 WO 97/OOg67 ~ PCT/US96/10729 diaza-s-in~ce~e-3-propionic acid), BODIPY~ 581/691 (4,~difluoro-5-(4-phenyl-1,3-bllt~tlie~yl)-4-bora-3a,4a-diaza-s-indacene-3-propioI-i~ci(l)~nd BODIPY~ 589/616 (6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phe~o~y)acetyl)amino)hexanoic acid).
As used herein, "DNA seqllencin~' refers to the process of determinin~ the nucleic acid sequence of a DNA strand of interest.
As used herein "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 fr~gment~ as they move through the gel. "Automated DNA
sequencer" refers to the instrument which is able to perform automated DNA sequencing.
As used herein, "seqlle~cing primer" means a synthetic oligonucleotide, restriction fr~gment enzymatically-synt.hesi7.ed DNA
fragment, or the like which hybridizes specifically to a region ~ x;..~te to the region of DNA to be sequenced. "Universal seqlla~cing 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 kno~,vn in the art are -21M13, M13-40 and -36M13.
As used herein, "5' position" refers to the 5' position on the deoxyribose moiety of a polynucleotide.
As used herein, "3' position" refers to the 3' position on the 26 deo~yribose moiety of a nucleotide.
As used herein, "base ~tt~chmant" or "dye-terminator" refers to a molecule, particularly a fluorescent dye, ~tt~chad to the C7 position of a purine termin~t.ing base or the C5 of a pyrimidine termin~ting base.
As used herein, "AP-3" or "AP-3 nucleotide" refers to the 3-amino-1-~1 o~y~lyl linker ~tt~he~ to the 5 position of pyrimidines or the 7 position of 7 ~le~7~purines. See Figure 7.

CA 0222~31 1997-12-22 WO 97/00~67 PCT/US96/10729 As used herein, "BODIPY'D linker" or "BODIPY~ functional group"
refers to a sub~ uled or unsubstituted alkyl cont~ining one to thirty carbons and at least one functional group. Two different BODIPY~
linkers are illustrated in Figure 1.
As used herein, "FAM" shall refer to 5-carboxy-fluorescein, "JOE"
shall refer to 2',7'-dimetho~y-4',5'-dichloro-6-carboxy-fluorescein, "TAMRA" shall refer to N,N,N',N'-tetramethyl-6-carboxy-rho~l~mine, "ROX" shall refer to 6-carboxy-X-rho~l~min-a.
As used herein, electrophoresis "lanes" or "tracks" or "columns"
refers to the particular path in the electrophoretic medium in which the sequencing products are run. For example, the sequencing products ter~nin~t.in~ in dideoxy,~r~eIlo.~ina, dideoxycytodine, dideoxyguanosine or dideogythy~ni(lina may be run in four separate lanes, or, if labeled differentially, in the same lane.
As used herein, "linkers" or "linker arms" refers to molecules that tether a dye to a primer. Typical linker molecules include ~lk,~nes of various lengths.
As used herein, "automated GeneScanner" refers to an instrument capable of performing analysis of fluorescently-labeled DNA or RNA.
~0 As used herein, "Taqman" or "T~qm~n assay" refers to assays that utilize the 5' - 3' exonllcle~e activity of Taq polymerase in a polymerase chain reaction to generate a specific detectable signal concomit~nt.ly with amplific~t.ion. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and ~esignad to hybridize within the target sequence, is introduced into the polymerase chain reaction assay.
~nna~lin~ of the probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5' - 3' e~onuclease 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 iml,~ ovel~lent over more cumbersome detection CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 methods. In one such assay, the oligonucleotide that is degraded has at least two light-emi~in~ fluorophores attached. The fluorophores interact each other to modify (quench) the light ami~ion of the fluorophores. The 5'-most fluorophore is the quencher fluorophore. The 3'-most fluorophore is the quenched fluorophore. In another type of Taqman assay, 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 emi~ion of the label.
As used herein, "labeled oligonucleotide" refers to the oligonucleotide in the T~qm~n assay that is labeled with at least two BODIPY~ fluorophores.
As used herein, "quenched" refers to the interaction of the at least two BODIPY0 fluorophores on the labeled oligonucleotide wherein when both BODIPY~' fluorophores are present on the labeled oligonucleotide, fluorescence of either fluorophore is not detected.
As used herein, "quencher fluorophore" refers to the BODIPYa' fluorophore at a position most 5' on the labeled oligonucleotide.
As used herein, "quenched fluorophore" refers to the BODIPY~
fluorophore at a position most 3' on the labeled oligonucleotide.
As used herein, "qllenc~er agent" refers to interc~l~ting compounds and the like simil~r to ethydium bromide for use in a Taqman assay fiimil~r to that used in the method of Fisher, et al., U.S. Pat. No.
5,491,063.
2~ One novel aspect of the present invention is to provide 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, the method comprising the steps of: forming a mixture of a first, a second, a third, and a fourth clas~ of polynucleotides, each polynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled at the 5' -CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 position with a ~lrst fluorophore; each polynucleotide in the second class having a 3'-terminal dideo~y~idine and being labeled at the 5' position with a second fluorophore; each polynucleotide in the third class having a 3'-terminal dideo2~yguanosine and being labeled at the 6' position with a third fluorophore; and each polynucleotide in the fourth class having a 3'-terminal dideoa ythymidine and being labeled at the 5' position with a fourth fluorophore; wherein at least one of said fluorophores is a BODIPY0 fluorophore, and, wherein if said first, second, third and fourth fluorophores are all different, said polynucleotides can be electrophoresed in a same or a di~erent lane; or wherein if any of said first, ~econd, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;
electrophoretically separating on a gel by size the polynucleotides;
~ min~t.inf~ with an illllmin~t.ion beam the bands on the gel, the illllmin~tion beam. being capable of c~ ing the fluorophores to fluoresce;
and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.
Another aspect of the present invention allows BODIPY0 fluorophores to be used in comhin~t.;on with prior art fluorophores and commercially-available so~w~e. This method involves disting~ hing polynucleotides having different 3'-terminal dideoxynucleotides in the chain termination method of DNA sequencing, the method co~ ising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the ~lrst class having a 3'-terminal dideoxyadenosine and being labeled at the ~' position with BODIPY'9 523/547, BODIPY~ 530/650 or JOE; each polynucleotide in the second class having a 3'-terminal dideu~y~ idine and being labeled at the 5' position with BODIPY0 503/512 or FAM; each polynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled at the 5' position with BODIPY~' 558/668, BODIPY0 564/570 or TAMRA; and each polynucleotide in the fourth class having a 3'-terminal CA 0222~31 1997-12-22 WO 97/00!J67 PCT/US96/10729 dideoxythymi~ina and being lsbeled at the 5' position with BODIPY~
581/591, BODIPY'19 589/616 or ROX; wherein at least one of the classes is labeled with a BODIPY~ fluorophore; electrophoretically separating on a gel by size the polynucleotides; illllmin~t.ing with an illllmin~t.ion beam the bands on the gel, the illllmin~t.ion beam being capable of c~ ing the fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.
In another aspect of the present in~ tion, there is provided a method of disting~ hing polynucleotides having different 3'-terminal dideo~llucleotides in the chain termin~t.ion method of DNA sequencing, the method co~ ising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled at the 5' position with a first BODIPY~ fluorophore; each polynucleotide in the second class having a 3'-terminal dideo~y~idine and being labeled at the 5' position with a second BODIPY~' fluorophore; each polynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled at the 5' position with a third BODIPY0 fluorophore; and each polynucleotide in the fourth class having a 3'-terminal dideoxythymidine and being labeled at the 5' position with a fourth BODIPY~ fluorophore;
wherein said first, second, third and fourth BODIPY0 fluorophores are all different; electrophoretically separating on a gel by size the polynucleotides; illllmin~ting with an illllmin~tion beam bands of said gel, said illllmin~tion beam being capable of c~ ing said BODIPY~' fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the dyes.
In a preferred embodiment, said BODIPY~ fluorophores have an adsorption m~im~ of about 450 to 700, and an emission m~im~ of about 450 to 700. In a more preferred embodiment, said BODIPY0 fluorophores have adsorption m~im~ of about 500 to 625, and an emission m~im~ of about 600 to 625.

CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 In one aspect of the present invention, said 3'-terminal dideoxyadenosine is labeled at the 6' position wi~h BODIPY~ 523/547;
said 3'-terminal dide-~y~;ylidine is labeled at the 5' position with BODIPY~ 503/l;12; 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-availLable software. Howt~vt~-, it should be clear that one ~killed in the art of co~ u~er software design that software could be altered such that the software could read different BODIPY~D dyes attached to different classes of polynucleotides by way of different linker arm chemistries.
In a preferred embodiment, said chain termin~t.iQn method of DNA
sequencing is performed by an automated DNA sequencing instrllmen~.
In another preferred embodiment, the method of the present invention further ~ncludes the step of exte~in~ from a primer a plurality of polynucleotides by means of a DNA polymerase suitable for DNA
sequencing or a reverse tran~criptase 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.
In another preferred embodiment of the present invention, said DNA polymerase is selected from the group of ThermoSequenase, Klenow fr~rneI-t, SequenaseX, Bst DNA polymerase, AmpliTaq~ DNA polymerase, Pfu(exo-)DNA polymerase, rTth DNA polymerase or Vent(exo-)~DNA
polymerase, and said reverse transcriptase is selected from the group of AMV-RT or M-MuLV-RT. In the case of RNA, RNA polymerase is used.
In another embodiment of the present invention, said BODIPYX
~ fluorophores are coupled to a primer suitable for sequencing by linkers.$0 In a more preferred embodiment of this aspect of the present invention, said linker arms are selected from the group of (CH2)3 (CH2)G, and (CH2),2 CA 0222~31 1997-12-22 In yet another aspect of the present invention, said polynucleotide i8 labeled with more than one fluorophore, wherein said fluorophores include at least one BODIPY~' fluorophore and at least one additional fluorophore. In a more y~af.dr~ed embo~ime~t. of this aspect of the invention, said ~ tional fluorophore has an adsorption m~im~ of about 475 to about 650. In another embodiment of this aspect of the present invelltior, said ~(l(litional fluorophore is a BODIPY~ fluorophore or FAM.
In an additional aspect to the present invention, methods are provided for the use of BODIPY0 fluorophores for DNA seqllencing wherein the BODIPY~ fluorophore is ~tts~.hetl at the 5' end of the products of the seqllencing 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 seqllencing reaction.
In another aspect of the present invention there is provided a method for disting~ ing polynucleotide sequences in a hybridization method of DNA sequencing, said method comprising the steps of:
syntl~e~ in~ 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 deu~y~idine at said position and is labeled at the 5' position with a second fluorophore; each oligonucleotide in the third class having a deoxyguanosine at said position and is labeled at the 5' position with a third fluorophore; and each oligonucleotide in the fourth class has a deoxythymidine at said position and is labeled at the 5' position with a fourth fluorophore; wherein at least one of said fluorophores is a BODIPY~ fluorophore; hybridizing said oligonucleotides to a single-stranded DNA target immobilized to a solid support, wherein said solid CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 support i8 in a grid format, to form a hybridized product; washing said hybridized product to remove any unhybridized oligonucleotide or target;
illllmin~tin~ with an illllmin~t.ion beam the solid support, said illllmin~tion beam being capable of c~ in~ said BODIPY0 fluorophores to fluoresce;
and identif ying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the dyes.
In an important aspect of the present invention, there is provided a method for genetic analysis of DNA fragments wherein said DNA
fr~nants are labelled vrith at least one BODIPY'19 fluorophore.
Another important and novel aspect of the present invention i8 to provide a method for distinguishing polynucleotides having different 3'-terminal dideoxyribonucleotides in any method of chain termination DNA
seqlla~r.in~, the method comprising the steps of: forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having a 3'-terminal dideoxyadenosine triphosphate, said 3'-terminal dideoxy~(le~ofiine triphosphate being ~tt~ch~d at the 7 position of the 7-deazapurine to a 3-~mino~ lo~y~lyl linker, said linker then Att~chad to a BODIPY6' linker at a 3 position of a first BODIPY~ fLuorophore that contains at least one reactive functional group; each polynucleotide in the second class having a 3'-terminal dideo~y~iy~idine triphosphate, said 3'-terminal dideo~y.;ylidine triphosphate being ~tt~clle~ at the 5 position of the pyrimidine to a 3-amino-l-~lo~yllyl linker, said linker then attached to a BODIPYa9 linker at a 3 position of a second BODIPY~ fluorophore that contains at least one reactive functional group; each polynucleotide in the third class having a 3'-terminal dideoxyguanosine triphosphate, said 3'-terminal dideo~yguanosine triphosphate being attached at the 7 position of the 7-~e~ rurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY~ linker at a 3 position of a third BODIPY~9 fluorophore that contains at least one reactive functional group; each polynucleotide in the fourth class having a 3'-terminal dideoxythymidine triphosphate, said 3'-CA 0222~31 1997-12-22 WO 97/OOg67 PCI'/US96/10729 terminal dideoxythymidine triphosphate being ~tt~ch~d at the 5 position ofthe pyrimitlina to a 3-amino-1-~1 o~y~lyl linker, said linker then ~ched to a BODIPY~ linker at a 3 position of a fourth BODIPY'!D fluorophore that contains at least one reactive functional group; wherein if said first, ~i second, third and fourth BODIPY~ fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane; or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes; electrophoretically separating on a gel by size the polynucleotides; illl.min~t.ing with an illllmin~t.ion beam the bands on the gel, the illllmin~t.icn beam being capable of c~ ing the fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluoresce~ce or absorption spectrum of the fluorophores.
Yet another embodiment of the present invention provides for the 1~ method of ~i~tin~li~hing 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 polynucleotide in the first class having an ~ o~ina triphosphate, said ~lerlosina triphosphate being ~tts~ch~d at the 7 position of the 7-deazapurine to a 3-amino-1-~1o~y~lyl linker, said linker then ~tt~ched to a BODIPY~ linker at a 3 position of a first BODIPY~ fluorophore that contains at least one reactive functional group; each poly-nucleotide in the second class having a cytidine triphosphate, said cy-tidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-~1 o~y~lyl linker, said linker then attached to a BODIPY0 linker at a 3 position of a second BODIPY~ fluorophore that contains at least one reactive functional group; each poly-nucleotide in the third class having a guanosine triphosphate, said guanosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-l-propy-nyl linker, said linker then attached to a BODIPY9' linker at a 3 position of a third BODIPY0 fluorophore that contains at least one CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 reactive functional group; each polynucleotide in the fourth class having a uracil triphosphate, said uracil triphosphate being At.~D~hed at the 5 position of the pyrimidine to a 3-amino-1-1,l o~yllyl linker, said linker then ~tt~rhed to a BODIPY~ linker at a 3 position of a fourth BODIPY~
fluorophore that contains at least one reactive functional group; wherein if said first, second, third and fourth BODIPY~ fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane; or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes; electrophoretically separating on a gel by size the polynucleotides; illllmin~t.in~ with an ill~lmin~~ion beam. the bands on the gel, the illllmin~t.ion beam being c~p~hla of c~ inz the fluorophores to fluoresce; and identifying the cl~e~ of poly~ucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.
In another embodiment of the present invention, there is provided a method for (1i~t.in~li~hing polynucleotides having different deoxyribonucleotides in any method of labelling polynucleotides by enzymatic incorporation, the method comprising the steps of: forming a ~20 mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having a deoxyadenosine triphosphate, said deoxyadenosine triphosphate being ~tt~ch~d at the 7 position of the 7--~e~7.~purine to a 3-amino-1-propynyl linker, said linker then 7~tt~ch~d to a BODIPY'IP linker at a 3 position of a first BODIPY8' 2~; fluorophore that contains at least one reactive functional group; each polynucleotide in the second class having a deo~y~;ylidine triphosphate, said deo~y.;y~idine triphosphate being attached at the B position of the pyrimidine to a 3-amino-1-~ol.yllyl linker, said linker then ~t.t~ch~d to a ~ . BODIPY~ linker at a 3 position of a second BODIPY'~9 fluorophore that contains at least one reactive functional group; each poly-nucleotide in the third class having a deoxyguanosine triphosphate, said deoxyguanosine WO 97/OOg67 PCT/US96/10729 triphosphate being ~thched at the 7 position of the 7-deazapurine to a 3-amino-1-~lopyllyl linker, said linker then ~tt~che~ to a BODIPY~ linker at a 3 position of a third BODIPY0 fluorophore that contains at least one reactive functional group; each polynucleotide in the fourth class having a deo2~ythymi-1ine triphosphate, said deoythymidine triphosphate being ~tt~clled at the 5 position of the pyrim~ n~ to a 3-amino~ lo~ylyl linker, said linker then ~tt~ched to a BODIPY0 linker at a 3 position of a fourth BODIPY~ fluorophore that contains at least one reactive functional group; wherein if said ~lrst, second, third and fourth BODIPY~
fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane; or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;
electrophoretically separating on a gel by size the polynucleotides;
illllmin~ting with an illllmin~tion beam the bands on the gel, the illllmin~tion beam being capable of ~ ing the fluorophores to fluoresce;
and identifying the classes of polynucleotides in the bands by the flu~,lescellce or absorption spectrum of the fluorophores.
In one aspect of the present invention, said adenosine triphosphate, de~oyadenosine triphosphate or 3'-terminal dideoxyadenosine triphosphate is labeled with BODIPY'9 523/547 or BODIPY0 530/550; said cytidine triphosphate, deoxy~ idine triphosphate or 3'-terminal dideoxy~;~Lidine triphosphate is labeled with BODIPY0 576/689, BODIPY0 581/591, or BODIPY~9 589/616; said guanosine triphosphate, deoxyguanosine triphosphate or 3'-terminal dideoxyguanosine triphosphate is labeled with BODIPYX 503/512; and said uracil triphosphate, deoxythymidine triphosphate or 3'-terminal dideo~ythymidine triphosphate is labeled with BODIPY'!D 558/568 or BODIPY~ 564/570. Labelling 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 CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 that software could be altered such that the software could read different BODIPY0 dyes atl;ached to different classes of polynucleotides.
Another a;3pect of the present invention allows BODIPY'~9 fluorophores to be used in combination with prior art fluorophores and coInm~rcially-available software.
In another preferred embo-lim~nt, 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 ~lrst 4,4-difluoro-4-bora-3A,4A-diaza-s-in~ceIl~
(BODIPY~) fluorophore is a quencher fluorophore and a second 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY~I9) fluorophore is a qlleIlclled fluoropY~ore.
A preferred embodiment of this aspect of the present invention provides BODIP'Y~' 564/570 (4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid) as a quencher fluorophore.
An additional preferred embo~iment of the present invention providesBODIPY~576/589 (4,4-difluoro-6-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid) as a quencher fluorophore.
A further preferred embo-~ime~t of the present invention provides BODIPY~ 581/591 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-in~7~rçne-3-propionic acid) as a quencher fluorophore.
Another preferred embodiment of the present invention provides 2~; BODIPY~ 558/568 (4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-incl~rene-3-propionic acid) as a quencher fluorophore.
Yet a further preferred embodiment of the present invention provides BODIP~ 581/591 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid) as a quencher fluorophore.

CA 0222~31 1997-12-22 WO 97/00~67 PCT/US96/10729 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 ~1 efer~ ed embodiment of the present invention provides BODIPY~ 523/547 (4,4-difluoro-6-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,~difluoro-5,7-diphenyl-~bora 3a,4a diaza s inrl~r~n.9 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 T~qm~n assay, wherein said at least one 4,4-di~1uoro-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 qllench~d (BODIPY~) fluorophores mentioned above can be used.

The following ~Y~mple~ are offered by way of illustration and are not included to limit the invention in any manner. The examples show the procedures for syntll~si~ing BODIPY~-tagged primers and performing DNA sequencing with said primers.

~XAMPLE 1 BODIEJY~ Fluorophores can S~ e for C~).uv~l~Liomll Seq~lqncin~
Dyes To ~Y~mine the role of BODIPY~ dyes in automated DNA
26 sequencing, substitution experiments were performed by replacing a conventional dye with a corresponding BODIPY~ dye having simil~r absorption/emission m~im~ Different linker arms coupled to a universal sequencing primer were synthesi7ed to chemically alter BODIPY~I9 dye-WO 97/00~67 PCT/US96/10729 labeled primerx to mimic the gel mobility pattern of conventional dye-labeled primers. Thus, BODIPY~ dyes can replace one or more prior art dyes.

A. ~ Pnt~: DINA synthesis reagents were purchased from Applied Bio~y~ellls, Inc. (ABI) except 5'-amino-modifier C3, C6, and C12 phosphor~mklites were purchased from Glen Research. Oligonucleotides R865, R932, R930, and R931 (Figure 2) were synthesized tril~yl-on (0.2 ~mole scale) using either an ABI model 380B or model 394 DNA
~3yntl~e~i~er and purified using Nensorb~ 20 columns according to the manllf~rtllrer's protocol (du Pont de Nemours & Co.). FAM-NHS, JOE-NHS, TAMRA-NHS, and ROX-NHS ester were purchased from ABI. 5-FAM-SE and BODIPY'19-SE dyes were purchased from Molecular Probes and resuspended in anhydrous DMSO (50 mg/mL).

B. ~r~ ,iO~ of nu~l ~c~ r~.D. Purified R865 primer (1.0 ~Lmole) was resuspended in 240 ~LL of 0.5 M NaHCOJNa2CO3 (pH 9.0) buffer and divided into eight aliquots. To each tube, 3 ~L of either FAM-NHS ester, 5-FAM-SE, JOE-NHS ester, TAMRA-NHS ester, ROX-NHS ester, or 5 ~L
of BODIPY~ 503/512-SE, BODIPY~ 523/547-SE, BODIPY'9 530/550-SE, BODIPY'D 558/56i8-SE, BODIPY~ 564/570-SE, BODIPY~ 576/589-SE, BODIPY0 581/591-SE, or BODIPY~ 589/616-SE, was added. Purified R930, R931, or R932 primers (0.6 ~Lmole) were resuspended in 200 ~L of 0.5 M NaHCOJNa2CO~, pH 9.0 buffer and divided into seven aliquots. To each tube, 5 ~L of either BODIPY~ 503/512-SE, BODIPY~ 530/550-SE, BODIPY~ 558/568-SE, BODIPY~ 564/570-SE, BODIPY~ 576/589-SE, BODIPY~ 581/591-SE, or BODIPY~ 589/616-SE, respectively was added.
Reactions were incubated at 25~ C for 16 h. Following ethanol - precipitation, dye-labeled primers were purified by reverse-phase high performance liquid chromatography tRP-HPLC). Fluorescent primers CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 were resuspended in 10 mM Tris-HCl, pH 8.0 and lmM EDTA and diluted to 0.4 pmoV~L.

C. RP-HPLC purifi~ n of oli~~ -rl~L~ The RP-HPLC hardware system used consists of a Beckm~n 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. 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.

D. ~ lt~ BOD~Y~ lluu~v~hores can s~ ? for co-.v~ n~l seqnan~ nF d~e~ or fluorophores. The chemical structures of different fluorophores and their corresponding absorption/emission In~rim~ are shown in Figure 1. FAM, JOE, TAMRA, and ROX are four conventional fluorophores utilized in automated DNA sequencing. To eY~mine the role of BODIPY~' fluorophores in DNA sequencing, substitution experiments were performed replacing conventional dye-labeled primers with BODIPY~s that C.)l~ ond to the emission spectrum of the 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. Here, three dye-labeled termination products (i.e., FAM, TAMRA, and ROX) were generated, combined with either JOE or BODIPY~ 530/550 termin~tinn products, and analyzed by automated DNA
sequencing. With the exception of BODIPY~ 589/616 reactions, BODIPY

WO 97/00~67 PCTtUS96/10729 503/512-, BODIPY 523/547-, BODIPY- 530/550-, BODIPY' 558/568-, BODIPY 564/570-, BODIPY 576/589-, and BODIPY' 581/591-labeled terrnination products migrated apprn~im~tely 3/4 to 1 base pair faster through the gel th~an FAM-, JOE-, TAMRA-, or ROX-labeled termin~t.ion products, respectively. The discrepancy between the two reactions is the result of the altered mobility of the different dye structures.
Although ~oftware mo~ific~qtj-lns could have been employed to C~r~ dye-primer mobility shifts, ch~mic~l morlific~tion of the R865 primer was performed (Figure 2). Oligonucleotides R930, R931, and R932 were dye-labeled with the BODIPY~ dyes listed in Figure 1 and purified by RP-HPLC. A~ shown in Figure 3, increasing the linker arm length from (CH2)~ to (CH2)l2 (R932) or addition of one 5' base plus (CH2)3 (R930) or (CH2)6 (R931) linker arm lengths slowed the mobility of BODIPY~
503/512-, BODIPY~ 530/550-, and BODIPY~ 564/570-labeled ter-min~tion 1~ products. In fact, BODIPY~ 503/512-R930, labeled termination reactions mimicked the sp~cin~ pattern of FAM-R865, BODIPY~D 523/547-R931 and BODIPY~ 530/550-R930 mimicked the spacing pattern of JOE-R865, BODIPY~!9 558/568-R930 and BODIPY~ 564/570-R930 mimicked the spacing pattern of TAMRA-R865, and BODIPY~ 576/589-R931, BODIPY~!9 681/591-R930, and BODIPY'19589/616-R865 mimi~.k~d the spacing pattern of ROX-R865, respectively, (compare highliFhted boxes).

~XAMPLE 2 BODIPY0 Dyes do Ilot E~equire Dinr~r~ lll.ial Labeling or &n~w~lra Correc~ion for D~~ nri~ in Mobili1~y Additionally and particularly distinctively, the overwh~lmin~
majority of BODIPYa9 fluorophores alter the mobility of termin~tion - products zn the same way, thus nullifying the need for chemical alteration of the fluorophore or software correction to generate accurate, evenly-spaced DNA sequences. Thus, due to their improved spectral qll~lities, WO 97/OOg67 PCT/US96110729 the use of 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'19 fluorophores leads to loved automated DNA sequencing in particular.

A l~ pntQ: DNA synth~ re~nt~ were purchased from Applied Bio~y~ s, Inc. (ABI). Oligonucleotides were synthesi7e-1 trityl-on (0.2 ,umole scale) using either an ABI model 380B or model 394 DNA
synthesi7er and purified using NensorbTM 20 columns according to the manufacturer's protocol (du Pont de Nemours & Co.). BODIPY 523/547 propionic acid (PA), and all BODIPY-succinimidyl ester (SE) dyes were purch~e-l from Molecular Probes. BODIPY -SE dyes were resuspended in anhydrous DMSO (50 mglmL), and BODIPY-523/547-PA was converted to BODIPY 523/547-SE acco..lillg to the manufacturer's protocol.

B. Pr~rd~ionofflu~J ~.l,~ ~.n. PurifiedR930primers(0.4~mole) l~i was resuspended in 160 ~L of 0.5 ~I NaHCOJNa2CO3 (pH 9.0) buffer and divided into four aliquots. To each tube, 5 ~L of BODIPY- 503/512-SE, BODIPY- ~64/570-SE, or BODIPY- 581/591-SE was added. To the fourth tube, 35 ~L of 0.25 ~ NaHCOJNa2CO9, pH 9.0 buffer and 30 ~L BODIPY
523/547-SE were added. Reactions were incubated at 25~C for 16 h.
Following ethanol precipitation, dye-labeled primers were purified by . c,v~ De-phase high performance liquid chromatography (RP-HPLC).
Fluorescent primers were resuspended in deionized (D.I.) water and diluted to 0.4 pmoV~L.

C. RP-HPLC purifi~;on 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 WO 97/OOg67 PCI~/US96/10729 RP-300 column (4.6 mm x 250 mm) where "Buffer A" is 100 m triethyl~n~monium acetate (TEAA), pH 7.0, and "Buffer B" is 100 m~
TEAA, 70% (v/v) acetonitrile. Dye-labeled oligonucleotides were purified using the following gradient con~ n~: 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.

D. ~oknllt~: Different BODIPY~ dyes do not alter significantly gel mobility. The striking observation that the same linker arm modification was required to substitute BODIPY~I9 dyes for conventional dyes (see F.~mrle 1, above) led to the disco~/.a. ~ that BODIPY~ dyes could generate accurate, evenly-spaced DNA seqllencin~ data without software correction for discrep~n~ s in mobility. BODIPY~ 503/512-"C", BODIPY~
530/550-"A", and BODIPY~ 564/570-"G", and BODIPY~9 581/591-"T" were chosen based on their chemical structure similarity. Figure 4 shows the comparison of DNA sequencing reads generated from four conventional dye-primers and four BODIPY~ dye-primers using two different M13 clones. Figure 6 depicts the normalized emi~ n spectra of four conventional dye-primers and BODIPY~ dye-primers. It is important to note that all BODl[PY~' dyes were tethered to the primer via the tethers XO in Figure 2, and that no differential linker or nucleotide mo.lifi~tion was required to achieve a precise, evenly-spaced, easily-read sequence re~rling l;~XAMPLE 3 MPt~ul for BODIPY~ Ener~ Transfer (BET) primer~

To increase l;he emissiom intensity, doubly-labeled dye-primers were 2~ constructed and evaluated for fluoroescence energy transfer (ET). To achieve efficient ET and m~imim~l signal, 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 WO 97/OOg67 PCr/US96/10729 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.

nty DNA synthasi~ reagents were purchased from ABI except ~; 5'-amino-modifier C3, C6, and C12, and amino modifier C6 dT
phosphoramidites were purchased from Glen Research. Oligonucleotides FET and BET primers were synthesi7e~1 trityl-on, using either an ABI
model 380B or model 394 DNA synthe~i7er. BODIPY 523/547 propionic acid (PA), and all BODIPY--SE dyes were purchased from Molecular Probes. BODIPY-SE dye were resuspended in anhydrous DMSO (50 mg/mL), and BODIPY 523/547-PA was converted to BODIPY 523/547-SE
acco.1il,g to the manufacturer's protocol.

B. E'lu .~ l primers: The donor dye for the FET-3 primer (5'-FAM-T-GTAAAACGACGGCCAGT was synthesized (0.2 ~mole) using 1~ 6-FAM amidite and C6dT (T-) and was ethanol precipitated. The donor dye for the BET-3 primer (5'-NTT-GTAAAACGACGGCCAGT, was synt.he~i7ed (0.2 ,umole) using either C3 or C6 amino link (N) and C6dT
(T-) and resuspended in 200 ~L of 0.1 N NaOH. To BET-3 primer, 10 ~L
of BODIPY 503/512-SE was added, incubated at 26 C for 10 min., ethanol precipitated, incubated in 200 ,uL of 80% acetic acid for 20 min., and ethanol preciritste-l. Both FET-3 and BET-3 primers were each resuspended in 160 ~L of 0.25 M NaHCOJNa2CO3, pH 9.0 buffer and divided into four aliquots. To three tubes, 3 ~L of either BODIPY-503/512-SE, BODIPY 564/570-SE, or BODIPY 581/591-SE, respectively 26 was added. To the fourth tube, 35 ~L of 0.25 M NaHCO3/Na2CO3, pH 9.0 buffer and 30 ~L BODIPY 523/547-SE were added. All dye labeling reactions were incubated at 25 C for 16 h. Following ethanol precipitation, dye-labeled primers were purified by RP-HPLC, resuspended in 10 mM
Tris-HCl, pH 8.0 and 1 mMF.nTA, and diluted to 0.4 pmoV~L.

w097/oog67 PCT~Sg6/l0729 ~1 C. RP-HPLC: The RP-HPLChaldwale system consisted of a Beçkm~n model 127 gradient solvent module, a Rheodyne model 7125 injector, an Applied Bio:~y~ ,s (ABI) model 759A absorbance detector, and a Spectra-Physics model SP4600 DataJet integrator. Gradient RP-HPLC
fi was performed USiLlg an ABI aquapore RP-300 column (4.6 mm X250 mm) where "Buffer A" is 100 mM triethylammonium acetate (TEAA), pH 7.0 and "Buffer B" is 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 B, 5 min. at a flow rate of 1.0 mL per min.

D. li~ A three base separation between either the FAM donor (FET-3) or the BODIP~ 503-512 donor (BET-3) (Figure 2B), and acceptor dyes was observed to give the greatest signal enh~nceInent for BODIPY-564/570 and BODIP~ 581/591 dyes, consistent with F ~-T ~ RA and FAM-ROX dye pairs. How~vel,BET-3 dye primers showed considerably greater ET efffciencies and signal enhancements over FET-3 dye primers.
See Table 1.

CA 0222~3l l997-l2-22 WO 97/00967 PCT~US96/10729 Signal ~~nhslnr~ n~ ET ~ nri~
A~xeptor dye~ BET-3 FET-3 BET-3 FET-3 BODrPY 503/512 154% 80%
BODrPY 523/547 91~ 35% 99% 93%
5BODrPY 569/570 360% 200% 99~ 92%
BODIl~Y 581/591 540% 470% 98% 67%

For BODIPY- 503/512 and BODIPY- 523/547 acceptor dyes, BET-3 dye-primersshoweda~pl~xi..,~telythesamesignalstrengthcomparedtotheir single dye counterpart, but ~ignific~nt 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 minim~l enhancement~ of the normally stronger fluorescent dyes to produce a four dye-primer set with roughly balanced signal intensities.
The sen~ ivi~y of the complete BET-3 primer set was ~min~d 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 sen~i~ivi~y of BET-3 dye-primers enables the direct loading of sequencing reactions onto gels without a previously-required laborious concentration step.
The unprocessed fluorescent ~ign~l~ generated from BET-3 sequencing reactions demonstrates the benefits of the uniform mobility, properly-balanced signal oul~ s and improved spectral purity of the WO 97/OOg67 ~ PCT/US96/10729 - pre~ent method. The raw data from BET-3 reactions generates a DNA
seqlle~c;n~ pattern that is visually intelp~atable and agree~ well with the COr~ e~onding analyzed data. In contrast, no discernable sequence pattern could be detected from the unprocessed sign~l~ of conventional primers.
5 Figure 4.

~XAMPLE 4 lM~t l~ for Ph~hr r~mi~it e T ~h~lin~

A. Reagents: 6-Aminohexanol, 2-cyanoethyl N,N-diiso~l o~ylchlorophosphor~mitlite, N,N-dii~o~ o~ylethyl~mine, and all solvents were purchased fromAldrich. Amine-VN-phosphoramidite was purchased from CLONTECH. BODIPY~-SE dyes were purchased from Molecular Probes and resuspended in anhydrous DMSO (50 mglmL).
FAM-NHS was purchased from ABI.

B. Synt~ : The general synthe.~i~ for two different schemes (route I
and II) is outlined in Figure 5.
Route I: Compound [I]: 6-aminohexanol (1 g, 8.5 mmol) is dried by co-evaporation with pyridine (2 X 10 mL; HPLC grade) under reduced ~la~ule. Residual pyridine is removed by evacuation at 0.1 mm Hg for 2 hours. The solid in methylene chloride (20 mL) is dissolved, and while stirring, freshly dlstilled diiso~l o~ylethylamine (3 lmL, 17 mmol) is added.
To the solution, a solution of BODIPY~-SE (8.5 mmol) in methylene chloride (10 mL) is added through a dropping funnel under an inert atmosphere. After 30 min of stirring, the progress of the reaction is monitored by thin layer chromatography (TLC). The reaction is usually complete in 1 hour. When the reaction is complete, the reaction mixture i8 washed with 5% NaHCO3 solution (3 X 15 mL), followed by saturated NaCl solution (15 mL). After drying the methylene chloride solution over CA 0222~31 1997-12-22 WO 97/00~67 PCT/US96/10729 anhydrous Na2SO4, the solvent is evaporated on a rotary evaporator to a yellow oil.
Route I: Compound [II]: Dye-labeled heY~n~l (6 mmol) is dried -under high vacuum for 3 hours and dissolved in fre~hly distilled THF
(from sodium metal and benzophenone, 10 mL). Diis~ o~ylethylamine (1 mL, 6 mmol) i8 added and the solution i8 stirred at 0~ C for 10 minutes.
2-cyanoethyl N,N-diiso~ o~ylchloro-phosphoramidite (1.7 mL, 7.5 mmol) is added d~o~wise through a syringe under an argon atmosphere. The amine hydrochloride should precipitate within 5 minutes of ~ ion. The mixture should be stirred for 30 minutes at 0~ C and then stirred at room tempela~u~e for 1 hour. The progress of the reaction is monitored by TLC. When the reaction is complete, the amine hydrochloride is removed by filtering through a sintered glass funnel under argon and the solid is washed with dry THF (2 X 10 mL). The combined filtrate is evaporated to a viscous oil on a rotary evaporator. The viscous oil is then dissolved in argon-purged ethyl acetate and the solution is washed with ice-cold 5%
aqueous NaHCO~ solution (2 X 10 mL) followed by saturated NaCl (10 mL). The ethyl ~cet~te solution is dried over anhydrous Na2SO~, filtered, and the filtrate is concentrated to a yellow oil on a rotary evaporator.
Route II: Compounds [III] and [IVl: 2'-deoxyribosyl moiety [III]
(Smith et al., 1994) is dissolved in piperidine, DMF. To this solution, a solution of BODIPYE'-SE (8.~ mmol) in DMF (10 mL) is added under an inert atmosphere. Diisopropylethylamine (1 mL, 6 mmol) is added and the solution is stirred at 0~ C for 10 minutes. 2-cyanoethyl 2~; N,N-diiso~ ylchlorophosphoramidite (1.7 mL, 7.6 mmol) is then added d~op~Nise through a syringe under an argon atmosphere. The amine hydrochloride should precipitate within 6 minutes. This mixture is then stirred for 30 minutes at 0~C and at room temperature for 1 hour. The progress of the reaction is monitored by TLC. When the reaction is complete, amine hydrochloride is removed by filtering through a sintered glass funnel under argon and the solid is washed with dry THF (2 X 10 CA 0222~31 1997-12-22 WO 97/OOg67 PCI~/US96/10729 mL). The combined filtrate is evaporated to a viscous oil on a rotary evaporator and t~ne viscous oil is then dissolved in argon-purged ethyl acetate and washed with ice-cold 5% aqueous NaHCO3 solution (2 X 10 mL), followed by satul~ted NaCl (10 mL). The ethyl ~cehte solution is then dried over anllydlous Na2SO~, filtered, and concentrated to a yellow oil on a evaporate rotary evaporator.

C. Purification: Route I: Compound [I]: A glass column is packed with 100 g silica gel-60 using a mia~ture of methanol: ethyl acetate: methylene chloride: (0.5:6.0:93.5 v/v/v) cont~inin~ 1% pyridine. The yellow oil is dissolved in 10 mL of the above solvent mixture and the solution is loaded onto the column. A mixture of methanol ethyl acetate: dichloromethane (1:12:87 v/v/v) i8 used to elute the column and fractions are collected.
Each fraction is checked for absorbance at the absorption wavelength m~rimum of the BODIPY' dye. Pooled fractions are then evaporated on lfi a rotary evaporal;or and the residue is dried to constant weight on high vacuum.
Route I: Compound [II]: A glass column is packed with 50 g silica gel-60 using a mixture of methanol: ethyl acetate: methylene chloride:
(0.5:6.0:93.5 v/v/v) cont~ining 19~o pyridine. The silica column is washed with a one-column volume of 2~% ethyl ~cet~te in he~ne. The sample is dissolved in a minimum volume of 50% ethyl ~cet~te in h~Y~n~ and loaded onto the column. The column is then eluted with 25% ethyl acetate in h~Y~ne and fractions are collected. The fractions are monitored by TLC
(50% ethyl acetate in hexane). The product is detected by shortwave W, ~5 and the desired fractions are combined and concentrated under reduced pressure using a rotary evaporator.
Route II: Compounds [III] and [IVl: A glass column is packed with 50 g silica gel-60 using a mixture of methanol: ethyl acetate: methylene chloride: (0.5:6.0:93.5 vlv/v) cont~qining 1% pyridine. The silica column is washed with one column volume of 26% ethyl acetate in l~-qY~ne. The CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96/10729 g~mple is then dissolved in a minimum volume of 60% ethyl acetate in l~aY~ne and loaded onto the column. The column is eluted with 25% ethyl acetate in hP~n~ and fractions are collected. The fractions are monitored by TLC (~;0% ethyl acetate in h~Y~ne). The product is detected by sho. ~wave W. The desired fractions are collected and concentrated under reduced ~res~u~e using a rotary evaporator.

~XAMPLE 5 l~r~~rul for labelling AP~ nucleotides A. R~ b. 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 anLydr~ s DMSO (50 mg/mL).

B. FluOrc~c~.lt termin~t~r~: To AP-3 ribonucleotides, deoxyribonucleotides or dideoxynucleotides (0.1 ~mole), 30 ~L of 0.25 M
NaHCOJNa2CO3, pH 9.0 buffer was added followed by the addition of 5 ~LL of BODIPY-SE dyes. All dye labelling reactions were incubated at 25~ C for 16 h. Dye-labeled nucleotides were purified by RP-HPLC, evaporated to near dryness and diluted in 10 mM Tris-HCl, pH 8.0, 1 mM
EDTA.

C. RP~ The RP-HPLC hardware system consisted 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 OD-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. Dye-labeled ribonucleotides, deoxynucleotides or dideoxynucleotides were WO 97/OOg67 PCT/US96/10729 purified using the following gradient conditions: 0% B, 5 minutes; 0% B -40% B, 30 minutes; 40~o B - 100% B, 18 minutes; 100% B, 5 minute~ at a flow rate of 1.0 mL per minute.

li~XAl-'lPLE 6 6 T~h~ll~ l DNA for T~lm~n A~8ay A. Reagents: DNA synthesis reagents were purchased from ABI
except 5'-amino-modi~ler C3, C6, and C12 and amino modi~ler C6 dT
phosphoramidites and 3'-amino-modifier CPG were purchased from Glen Research. Oligonucleotides BET primers were synthesi7ed on 3'-amino-modifier CPG column trityl-on, auto-cleavage using either an BI model 380B or model 394 DNA synthesi7.çr. All BODIPY-SE dyes were purchased from Molecular Probes. BODIPY-SE dye were resuspended in an~yd.ous DMSO (50 mg/mL).

B. Fluu.~lli, ~r~ c.,D. The leader sequences for BET dye-primers are 5'-NTGTT* or 5'-NACGTTGT* followed by any primer sequence that is completely complemen~ry to the tsrget sequence. Primers were synt.~esi7ed (0.2 ~mole) using either C3 or C6 amino link tN) and C6dT
(T*) and resuspended in 400 ,ul of 0.01 N NaOH. To each tube, 10 ~l of BODIPY 503/512-$E, BODIPY 523/547 or BODIPY 530/550 was added, incllh~tetl at 25~C for 10 min., ethanol precipitated, incubated in 200 ~Ll of 80% acetic acid ~or 20 min., and ethanol precipitated. BODIPY primers were resuspended in 200 ~L of 0.25 M NaHCO3/Na2CO3, pH 9.0 buffer and 10 ,uL of either BODIPY 588/568-SE, BODIPY 564/570-SE, BODIPY
576/589-SE, BODIPY 581/591-SE, or BODIPY 589/616-SE, was added and the mixtures were incubated at 25~ C for 16 h. Following ethanol - precipitation, dye-labeled primers were purifiedby RP-HPLC, resuspended in deionized (DI) water, and diluted to 0.4 pmol/~l.

CA 0222~31 1997-12-22 WO 97/OOg67 PCT/US96tlO729 C. RP-HPLC. The RP-HPLC hardware system consisted 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 260 mm) where "Buffer A" is 100 mM triethylammonium acetate (TEAA), pH 7.0 and "Buffer B" is 100mM TEAA 70% (v/v) acetonitrile. Dye-labeled oligonucleotides were purified using the following gradient conditions:
20% B, 6 minutes; 20% B-40% B, 30 minutes; 40% B- 100% B, 18 minutes;
100% B, 5 minutes at a flow rate of 1.0 ml per minute.

One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The oligonucleotides, dyes, methods, procedures and te(hniques described 1~ herein are ~ese~ltly ~plc3cntative of the preferred embodiments, are intended to be P~empl~ry, and are not intended as limit~t.ions on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, and are encomp~e.l within the spirit of the invention or de~med by the scope of the appended claims. All references specifically cited herein are incorporated by reference.

Claims (54)

1. A method for DNA sequencing wherein polynucleotide products of said DNA sequencing are 5'-end-labelled with substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY R. fluorophore).

2. A method for distinguishing polynucleotides having different 3'-terminal dideoxynucleotides in any method of DNA sequencing requiring electrophoresis of 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 polynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled at a 5' position with a first fluorophore; each polynucleotide in the second class having a 3'-terminal dideoxycytidine and being labeled at a 5' position with a second fluorophore; each polynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled at a 5' position with a third fluorophore; and each polynucleotide in the fourth class having a 3'-terminal dideoxythymidine and being labeled at a 5' position with a fourth fluorophore; wherein at least one of said fluorophores is a BODIPY R. fluorophore, and, wherein if said first, second, third and fourth fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane, and wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;
electrophoretically separating on a gel by size the polynucleotides; illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.

3. A method for 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 polynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled at a 5' position with BODIPY ~ 523/547, BODIPY ~ 530/550 or JOE; each polynucleotide in the second class having a 3'-terminal dideoxycytidine and being labeled at a 5' position with BODIPY ~
503/512 or FAM; each polynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled at a 5' position with BODIPY ~ 558/568, BODIPY ~ 564/570 or TAMRA; and each polynucleotide in the fourth class having a 3'-terminal dideoxythymidine and being labeled at a 5' position with BODIPY R
576/589, BODIPY R 581/591, BODIPY R 589/616, or ROX; wherein at least one of said classes is labeled with a BODIPY R fluorophore;
electrophoretically separating on a gel by size the polynucleotides; illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.

4. A method for 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 polynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled at a 5' position with a first BODIPY ~ fluorophore ; each polynucleotide in the second class having a 3'-terminal dideoxycytidine and being labeled at a 5' position with a second BODIPY ~ fluorophore; each polynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled at a 5' position with a third BODIPY fluorophore; and each polynucleotide in the fourth class having a 3'-terminal dideoxythymidine and being labeled at a 5' position with a fourth BODIPY ~ fluorophore; wherein said first, second, third and fourth BODIPY ~ fluorophore 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.

5. The method of claims 1,2,3, or 4, wherein said BODIPY ~
fluorophores have an adsorption maxima of about 450 to 700, and an emission maxima of about 450 to 700.

6. The method of claims 1,2,3, or 4, wherein said BODIPY ~
fluorophores have adsorption maxima of about 480 to 650, and an emission maxima of about 480 to 650.

7. The method of claims 2,3, or 4, wherein said chain termination method of DNA sequencing is performed by an automated DNA
sequencing instrument.

8. The method of claims 2,3,or 4, further including the step of extending from a primer a plurality of polynucleotides by means of a DNA
polymerase or a reverse transcriptase in the presence of dideoxyadenosine triphosphate, dideoxycytosine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate to form said first, second, third, and fourth classes of polynucleotides.

9. The method of claim 8, wherein said DNA polymerase is selected from the group of Thermosequenase, Klenow fragment, Sequenase R., Bst DNA polymerase, AmpliTaq R. DNA polymerase, Pfu (exo-)DNA polymerase, rTth DNA polymerase or Vent(exo-) DNA polymerase, and the reverse transcriptase is selected from the group of AMV-RT or M-MuLV-RT.

10. The methods of claim 1,2,3, or 4, wherein said BODIPY R.
fluorophores are coupled to a primer suitable for sequencing by a linker.

11. The method of claim 10, wherein said linker has the formula (CH2)n, where n = 1- 30.

12. The method of claim 11, wherein said linkers are selected from the group of (CH2)3, (CH2)6, and (CH2)12.

13. The method of claim 1,2,3, or 4, wherein said BODIPY R.
fluorophore is attached at the 5' end of the products of the sequencing reaction and an additional fluorophore is attached at a 3' position of the product of the sequencing reaction or at one or more internal positions of the products of the sequencing reaction.

14. The method of claim 13, wherein said additional fluorophore is a BODIPY R. fluorophore.

15. The method of claim 13, wherein said additional fluorophore is FAM.

16. The method of claim 14, wherein said additional fluorophore has an adsorption maxima of about 475 to 650.

17. The method of claim 16 wherein said additional fluorophore is BODIPY R. 523/547.

18. The method of claim 16, wherein said additional fluorophore is BODIPY R. 503/512.

19. A method for distinguishing polynucleotide sequences in a hybridization method of DNA sequencing, said method 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 a 5' position with a first fluorophore;
each oligonucleotide in the second class has a deoxycytidine at said position and is labeled at a 5' position with a second fluorophore;
each oligonucleotide in the third class having a deoxyguanosine at said position and is labeled at a 5' position with a third fluorophore; and each oligonucleotide in the fourth class has a deoxythymidine at said position and is labeled at a 5' position with a fourth fluorophore; wherein at least one of said fluorophores is a BODIPY R. fluorophore;

hybridizing said oligonucleotides to a single-stranded DNA
target immobilized to a solid support, wherein said solid support is in a grid format, to form a hybridized product;
washing said hybridized product to remove any unhybridized oligonucleotide or target;
illuminating with an illumination beam the solid support, 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.

20. The method of claim 19 wherein said at least one BODIPY ~
fluorophore has an adsorption maxima of about 450 to 700, and an emission maxima of about 450 to 700.

21. The method of claim 20, wherein said at least one BODIPY ~
fluorophore has adsorption maxima of about 480 to 650, and an emission maxima of about 480 to 650.

22. A method for genetic analysis of DNA fragments wherein said DNA
fragments are labelled at a 5' position with at least one BODIPY ~
fluorophore.

23. A method for 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 polynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled at a 5' position with BODIPY ~ 523/547; each polynucleotide in the second class having a 3'-terminal dideoxycytidine and being labeled at a 5' position with BODIPY~ 503/512; each polynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled at a 5' position with BODIPY~ 564/570; and each polynucleotide in the fourth class having a 3'-terminal dideoxythymidine and being labeled at a 5' position with BODIPY~ 581/591; wherein at least one of said classes is labeled with a BODIPY~ fluorophore;
electrophoretically separating on a gel by size the polynucleotides; illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.

24. A method for 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 polynucleotide in the first class having a 3'-terminal dideoxyadenosine and being labeled at a 5' position with BODIPY~ 523/547; each polynucleotide in the second class having a 3'-terminal dideoxycytidine and being labeled at a 5' position with BODIPY~ 581/591; each polynucleotide in the third class having a 3'-terminal dideoxyguanosine and being labeled at a 5' position with BODIPY~ 503/512; and each polynucleotide in the fourth class having a 3'-terminal dideoxythymidine and being labeled at a 5' position with BODIPY~ 564/570; wherein at least one of said classes is labeled with a BODIPY~ fluorophore;
electrophoretically separating on a gel by size the polynucleotides; illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce; and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.

25. A method for distinguishing polynucleotides having different 3'-terminal dideoxynucleotides in any method of chain termination DNA
sequencing, said method comprising the steps of:
forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide 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-1-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 polynucleotide in the second class having a 3'-terminal dideoxycytidine triphosphate, said 3'-terminal dideoxycytidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY~ linker at a 3 position of a second BODIPY~ fluorophore that contains at least one reactive functional group; each polynucleotide in the third class having a 3'-terminal dideoxyguanosine triphosphate, said 3'-terminal dideoxyguanosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY~ linker at a 3 position of a third BODIPY~
fluorophore that contains at least one reactive functional group; each polynucleotide in the fourth class having a 3'-terminal dideoxythymidine triphosphate, said 3'-terminal dideoxythymidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY~ linker at a 3 position of a fourth BODIPY~ fluorophore that contains at least one reactive functional group; wherein if said first, second, third and fourth BODIPY~
fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane; or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;
electrophoretically separating on a gel by size the polynucleotides;
illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce;
and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.

26. The method of claim 25, wherein the BODIPY~ fluorophores are selected from the group of BODIPY~ 530/550; BODIPY~ 503/512;
BODIPY~ 564/570; BODIPY~ 589/616; BODIPY~ 581/591; BODIPY~
523/547; BODIPY~ 558/568; and BODIPY~ 576/589.

27. The method of claim 25, wherein said 3'-terminal dideoxyadenosine triphosphate is labeled with BODIPY~ 523/547 or BODIPY~ 530/550; said 3'-terminal dideoxycytidine triphosphate is labeled with BODIPY~
576/589, BODIPY~ 581/591, or BODIPY~ 589/616; said 3'-terminal dideoxyguanosine triphosphate is labeled with BODIPY~ 503/512; and said 3'-terminal dideoxythymidine triphosphate is labeled with BODIPY~
558/568 or BODIPY~ 564/570.

28. The method of claim 25, wherein said BODIPY~ fluorophores have an adsorption maxima of about 450 to 700, and an emission maxima of about 450 to 700.

29. The method of claim 25, wherein said chain termination method of DNA sequencing is performed by an automated DNA sequencing instrument.

30. The method of claim 25, wherein said classes of polynucleotides are formed using a DNA polymerase selected from the group of Klenow fragment Sequenase~, Bst DNA polymerase, AmpliTaq~ DNA polymerase, Pfu(exo-)DNA polymerase, Thermosequenase~, rTth DNA polymerase or Vent(exo-) DNA polymerase, and the reverse transcriptase is selected from the group of AMV-RT or M-MuLV-RT.

31. A method for distinguishing polynucleotides having different ribonucleotides in any method of labelling polynucleotides by enzymatic incorporation, said method comprising the steps of:
forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide 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-1-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 polynucleotide in the second class having a cytidine triphosphate, said cytidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY~
linker at a 3 position of a second BODIPY~ fluorophore that contains at least one reactive functional group; each polynucleotide in the third class having a guanosine triphosphate, said guanosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY~ linker at a 3 position of a third BODIPY~ fluorophore that contains at least one reactive functional group; each polynucleotide in the fourth class having a uracil triphosphate, said uracil triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY~ linker at a 3 position of a fourth BODIPY~ fluorophore that contains at least one reactive functional group; wherein if said first, second, third and fourth BODIPY~ fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane; or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;
electrophoretically separating on a gel by size the polynucleotides;
illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce;
and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.

32. The method of claim 31, wherein the BODIPY~ fluorophores are selected from the group of BODIPY~ 530/550; BODIPY~ 503/512;
BODIPY~ 564/570; BODIPY~ 589/616; BODIPY~ 581/591; BODIPY~
523/547; BODIPY~ 558/568; and BODIPY~ 576/589.

33. The method of claim 31, wherein said adenosine triphosphate is labeled with BODIPY~ 523/547 or BODIPY~ 530/550; said cytidine triphosphate is labeled with BODIPY~ 576/589, BODIPY~ 581/591, or BODIPY~ 589/616; said guanosine triphosphate is labeled with BODIPY~
503/512; and said uracil triphosphate is labeled with BODIPY~ 558/568 or BODIPY~ 564/570.

34. The method of claim 31, wherein said BODIPY~ fluorophores have an adsorption maxima of about 450 to 700, and an emission maxima of about 450 to 700.

35. The method of claim 31, wherein said internal labelling and distinguishing polynucleotides is performed by an automated GeneScanner.

36. A method for distinguishing polynucleotides having different deoxyribonucleotides in any method of labelling polynucleotides by enzymatic incorporation, said method comprising the steps of:
forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide 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-1-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 polynucleotide in the second class having a deoxycytidine triphosphate, said deoxycytidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY~ linker at a 3 position of a second BODIPY~
fluorophore that contains at least one reactive functional group; each polynucleotide in the third class having a deoxyguanosine triphosphate, said deoxyguanosine triphosphate being attached at the 7 position of the 7-deazapurine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY~ linker at a 3 position of a third BODIPY~ fluorophore that contains at least one reactive functional group; each polynucleotide in the fourth class having a deoxythymidine triphosphate, said deoxythymidine triphosphate being attached at the 5 position of the pyrimidine to a 3-amino-1-propynyl linker, said linker then attached to a BODIPY~ linker at a 3 position of a fourth BODIPY~ fluorophore that contains at least one reactive functional group; wherein if said first, second, third and fourth BODIPY~ fluorophores are all different, said polynucleotides can be electrophoresed in a same or a different lane; or wherein if any of said first, second, third or fourth fluorophores are the same, said polynucleotides labeled with said same fluorophores are electrophoresed in separate lanes;
electrophoretically separating on a gel by size the polynucleotides;

illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the fluorophores to fluoresce;
and identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the fluorophores.

37. The method of claim 36, wherein the BODIPY~ fluorophores are selected from the group of BODIPY~ 530/550; BODIPY~ 503/512;
BODIPY~ 564/570; BODIPY~ 589/616; BODIPY~ 581/591; BODIPY~
523/547; BODIPY~ 558/568; and BODIPY~ 576/589.

38. The method of claim 36, wherein said deoxyadenosine triphosphate is labeled with BODIPY~ 523/547 or BODIPY~ 530/550; said deoxycytidine triphosphate is labeled with BODIPY~ 576/589, BODIPY~ 581/591, or BODIPY~ 589/616; said deoxyguanosine triphosphate is labeled with BODIPY~ 503/512; and said deoxythymidine triphosphate is labeled with BODIPY~ 558/568 or BODIPY~ 564/570.

39. The method of claim 36, wherein said BODIPY~ fluorophores have an adsorption maxima of about 450 to 700, and an emission maxima of about 450 to 700.

40. The method of claim 36, wherein said internal labelling and distinguishing polynucleotides is performed by an automated GeneScanner.

41. The method of claim 36, wherein said classes of polynucleotides are formed using a DNA polymerase selected from the group of Klenow fragment, Sequenase~, Bst DNA polymerase, AmpliTaq~ DNA polymerase, Pfu(exo-)DNA polymerase, Thermosequenase~, rTth DNA polymerase or Vent(exo-) DNA polymerase, and the reverse transcriptase is selected from the group of AMV-RT or M-MuLV-RT.

42. 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.

43. The oligonucleotide of claim 42, wherein said quencher fluorophore isBODIPY~564/570 (4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid)

44. The oligonucleotide of claim 42, wherein said quencher fluorophore is BODIPY~ 576/589 (4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid).

45. The oligonucleotide of claim 42, wherein said quencher fluorophore is BODIPY~ 581/591 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid).

46. The oligonucleotide of claim 42, wherein said quenched fluorophore is BODIPY~ 503/512-SE (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid).

47. The oligonucleotide of claim 42, wherein said quenched fluorophore is BODIPY~ 558/568 (4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid).

48. The oligonucleotide of claim 42, wherein said quenched fluorophore isBODIPY~589/616(6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid).

49. The oligonucleotide of claim 42, wherein said quenched fluorophore is BODIPY~ 523/547 (4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid).

50. The oligonucleotide of claim 42, wherein said quenched fluorophore is BODIPY~ 530/550 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid).

51. An oligonucleotide substituted with at least one 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY~) fluorophore 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.

52. The oligonucleotide of claim 51, wherein said quenched fluorophore isBODIPY~589/616(6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid).

53. The oligonucleotide of claim 51, wherein said quenched fluorophore is BODIPY~ 523/547 (4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid).

54. The oligonucleotide of claim 51, wherein said quenched fluorophore is BODIPY~ 530/550 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid).