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Description  |
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FIELD OF THE INVENTION
The invention relates generally to methods for determining the sequence of
a nucleic acid, and more particularly, to use of rhodamine dyes to
identify similarly sized DNA fragments separated by gel electrophoresis.
BACKGROUND
The ability to determine DNA sequences is crucial for understanding 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
antiparallel orientation by hydrogen bonds between complementary bases of
the nucleotides of the two strands:
deoxyadenosine (A) pairs with thymidine (T) and
deoxyguanosine (G) pairs with deoxycytidine (C).
Presently there are two basis approaches to DNA sequence determination: the
dideoxy chain termination method, e.g. Sanger et al, Proc. Natl. Acad.
Sci., Vol. 74, pgs. 5463-5467 (1977); and the chemical degradation method,
e.g. Maxam et al, Proc. Natl. Acad. Sci., Vol. 74, pgs. 560-564 (1977).
The chain termination method has been improved in several ways, and serves
as the basis for all currently available automated DNA sequencing
machines, e.g. Sanger et al, J. Mol. Biol., Vol. 143, pgs. 161-178 (1980);
Schreier et al, J. Mol. Biol., Vol. 129, pgs. 169-172 (1979); Smith et al,
Nucleic Acids Research, Vol. 13, pgs. 2399-2412 (1985); Smith et al,
Nature, Vol. 321, pgs. 674-679 (1987); Prober et al, Science, Vol. 238,
pgs. 336-341 (1987), Section II, Meth. Enzymol., Vol. 155, pgs. 51-334
(1987); Church et al, Science, Vol. 240 pgs. 185-188 (1988); and Connell
et al, Biotechniques, Vol. 5, pgs. 342-348 (1987).
Both the chain termination and chemical degradation methods require the
generation of one or more sets of labeled DNA fragments, each having a
common origin and each terminating with a known base. The set or sets of
fragments must then be separated by size to obtain sequence information.
In both methods, the DNA fragments are separated by high resolution gel
electrophoresis. In most automated DNA sequencing machines, fragments
having different terminating bases are labeled with different fluorescent
dyes, which are attached either to a primer, e.g. Smith et al (1987, cited
above), or to the base of a terminal dideoxynucleotide, e.g. Prober et al
(cited above). The labeled fragments are combined and loaded onto the same
gel column for electrophoretic separation. Base sequence is determined by
analyzing the fluorescent signals emitted by the fragments as they pass a
stationary detector during the separation process.
Obtaining a set of dyes to label the different fragments is a major
difficulty in such DNA sequencing systems. First, it is difficult to find
three or more dyes that do not have significantly overlapping emission
bands, since the typical emission band halfwidth for organic fluorescent
dyes is about 40-80 nanometers (nm) and the width of the visible spectrum
is only about 350-400 nm. Second, even when dyes with non-overlapping
emission bands are found, the set may still be unsuitable for DNA
sequencing if the respective fluorescent efficiencies are too low. For
example, Pringle et al, DNA Core Facilities Newsletter, Vol. 1, pgs. 15-21
(1988), present data indicating that increased gel loading cannot
compensate low fluorescent efficiencies. Third, when several fluorescent
dyes are used concurrently, excitation becomes difficult because the
absorption bands of the dyes are often widely separated. The most
efficient excitation occurs when each dye is illuminated at the wavelength
corresponding to its absorption band maximum. When several dyes are used
one is often forced to make a trade off between the sensitivity of the
detection system and the increased cost of providing separate excitation
sources for each dye. Fourth, when the number of differently sized
fragments in a single column of a gel is greater than a few hundred, the
physiochemical properties of the dyes and the means by which they are
linked to the fragments become critically important. The charge, molecular
weight, and conformation of the dyes and linkers must not adversely effect
the electrophoretic mobilities of closely sized fragments so that
extensive band broadening occurs or so that band positions on the gel
become reversed, thereby destroying the correspondence between the order
of bands and the order of the bases in the nucleic acid whose sequence is
to be determined. Finally, the fluorescent dyes must be compatible with
the chemistry used 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 interfer 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 automated 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; and Connell et al (cited above).
In view of the above, many analytical and diagnostic techniques, such as
DNA sequencing, would be significantly advanced by the availability of new
sets of fluorescent dyes (1) which are physiochemically similar, (2) which
permit detection of spacially overlapping target substances, such as
closely spaced bands of DNA on a gel, (3) which extend the number of bases
that can be determined on a single gel column by current methods of
automated DNA sequencing, (4) which are amenable for use with a wide range
of preparative and manipulative techniques, and (5) which otherwise
satisfy the numerous requirements listed above.
SUMMARY OF THE INVENTION
The invention is directed to a method of DNA sequencing wherein
electrophoretically separated DNA fragments having different 3'-terminal
nucleotides are identified by different rhodamine dyes. In particular, the
invention includes the rhodamine-labeled nucleotides defined below and
their use in the method of the invention. The invention is based in part
on the discovery of a set of spectrally resolvable rhodamine dyes linked
to chain-terminating nucleotides that are readily incorporated into
growing DNA chains by DNA polymerases, particularly Taq DNA polymerase. As
used herein the term "chain terminating nucleotide" refers to a nucleotide
or analog thereof which prevents further polynucleotide chain elongation,
or extension, after it has been incorporated into a growing DNA chain by a
DNA polymerase. Usually, the chain terminating property of such
nucleotides is due to the absence or modification of the 3' hydroxyl of
the sugar moiety. Preferably, the chain-terminating nucleotides are
2',3'-dideoxynucleotides.
As used herein the term "spectrally resolvable" in reference to a set of
dyes means that the fluorescent emission bands of the dyes are
sufficiently distinct, i.e. sufficiently non-overlapping, that target
substances to which the respective dyes are attached, e.g.
polynucleotides, can be distinguished on the basis of the fluorescent
signal generated by the respective dyes by standard photodetection
systems, e.g. employing a system of band pass filters and photomultiplier
tubes, or the like, as exemplified by the systems described in U.S. Pat.
Nos. 4,230,558, 4,811,218, or the like, or in Wheeless et al, pgs. 21-76,
in Flow Cytometry: Instrumentation and Data Analysis (Academic Press, New
York, 1985).
Whenever the rhodamines of the invention are used to label 3'-terminal
nucleotides, an important feature of the invention is the virtually
equivalent contributions to electrophoretic mobility of the DNA fragments
made by the four dyes. This result is unexpected because it is believed
that conformational interactions between the label and the adjacent
nucleotides are primarily responsible for the variability in
electrophoretic mobility of labeled DNA fragments. It was expected that
dye-labeled primers would always cause less variability in electrophoretic
mobilities since the dye would always interact with the same sequence of
nucleotides. However, part of the present invention is the discovery that
fragments with 3'-labeled nucleotides have much less variability in
electrophoretic mobilities than equivalent fragments with dye-labeled
primers.
DETAILED DESCRIPTION OF THE INVENTION
The spectrally resolvable set of rhodamine dyes of the invention consists
of tetramethylrhodamine, rhodamine X, rhodamine 110, and rhodamine 6G,
which are defined by Formulas I-IV, respectively. Throughout, the Colour
Index (Association of Textile Chemists, 2nd Ed., 1971) carbon numbering
scheme is used, i.e. primed numbers refer to carbons in the xanthene
structure and unprimed numbers refer to carbons in the 9'-phenyl.
##STR1##
wherein:
A is a group, such as carboxyl, sulfonyl, or amino, that may be converted
into a linking functionality; and
B is an anionic acidic group, preferably carboxyl or sulfonyl, and most
preferably carboxyl.
Preferably, the rhodamine-labeled chain-terminating nucleotides of the
invention have the following form:
XTP--L--R
wherein XTP is a chain-terminating nucleoside triphosphate; R is a
rhodamine dye selected from the group consisting of tetramethylrhodamine,
rhodamine X, rhodamine 110, and rhodamine 6G; and L is a linking group
between the base of the nucleoside triphosphate and the rhodamine dye.
XTP is an analog of the natural nucleoside triphosphate substrate of the
DNA polymerase employed which prevents further chain elongation after
incorporation. Several such analogs are available for each of the four
natural nucleoside triphosphates, e.g. Hobbs et al (cited above) gives a
list. Preferably, XTP is a 2',3'-dideoxynucleoside triphosphate. More
preferably, XTP is selected from the group consisting of
2',3'-dideoxy-7-deazaadenosine triphosphate, 2',3'-dideoxycytidine
triphosphate, 2',3'-dideoxy-7-deazaguanosine, 2',3'-dideoxyuridine
triphosphate, and 2',3'-dideoxy-7-deazainosine triphosphate. As used
herein, the term "dideoxynucleoside" includes nucleoside analogs whose
sugar moieties are either cyclic or acyclic. Conventional numbering is
used whenever specific carbon atoms of a base or sugar of a nucleoside are
referred to, e.g. Kornberg, DNA Replication (Freeman, San Francisco,
1980).
L can take on a number of different forms such that the length and rigidity
of the linkage between the dideoxynucleotide and the dye can vary greatly.
For example, several suitable base labeling procedures have been reported
that can be used with the invention, e.g. Gibson et al, Nucleic Acids
Research, Vol. 15, pgs. 6544-6467 (1987); Gebeyehu et al, Nucleic Acids
Research, Vol. 15, pgs. 4513-4535 (1987); Haralambidis et al, Nucleic
Acids Research, Vol. 15, pgs. 4856-4876 (1987); and the like. Preferably,
L is formed by reacting an N-hydroxysuccinimide (NHS) ester of a dye of
the invention with an alkynylamino-derivatized base of a
dideoxynucleotide. In this case, L is taken as the moiety between (1) the
5- or 6-carbon of the rhodamine and (2) the carbon of the base to which
the rhodamine is attached. Preferably, L is 3-carboxyamino-1-propynyl. The
synthesis of such alkynylamino-derivatized dideoxynucleotides of cytosine,
thymine, and adenine is taught by Hobbs et al in European patent
application No. 87305844.0 and Hobbs, J. Org. Chem., Vol. 54, pg. 3420
(1989), which are incorporated herein by reference. Briefly, the
alkynylamino-derivatized dideoxynucleotides are formed by placing the
appropriate halodideoxynucleoside (usually 5-iodopyrimidine and
7-iodo-7-deazapurine dideoxynucleosides as taught by Hobbs et al (cited
above)) and Cu(I) in a flask, flushing with Ar to remove air, adding dry
DMF, followed by addition of an alkylamine, triethylamine and Pd(0). The
reaction mixture can be stirred for several hours, or until thin layer
chromatography indicates consumption of the halodideoxynucleoside. When an
unprotected alkynylamine is used, the alkynylamino-nucleoside can be
isolated by concentrating the reaction mixture and chromatographing on
silica gel using an eluting solvent which contains ammonium hydroxide to
neutralize the hydrohalide generated in the coupling reaction. When a
protected alkynylamine is used, methanol/methylene chloride can be added
to the reaction mixture, followed by the bicarbonate form of a strongly
basic anion exchange resin. The slurry can then be stirred for about 45
minutes, filtered, and the resin rinsed with additional methanol/methylene
chloride. The combined filtrates can be concentrated and purified by
flash-chromatography on silica gel using a methanol-methylene chloride
gradient. The triphosphates are obtained by standard techniques.
Synthesis of the alkynylamine-derivatized dideoxyguanosine according to the
above reference requires specially modified quanine precursor
(6-methoxy-2-methylthio-7-deazapurine, X), which is obtained from the
starting material, 6-hydroxy-2-methylthio-7-diazapurine, XX. Conversion of
XX to 6-chloro-2-methylthio-7-deazapurine, XXX, according to Robins and
Noell (J. Heterocyclic Chem., Vol. 1, pg. 34 (1964)), followed by
displacement of the chloro substituent with methoxide (sodium salt in
refluxing methanol) yields X:
##STR2##
Preferably, the rhodamine NHS esters are synthesized in accordance with the
teachings of U.S. patent application No. 06/941,985. Important features of
the method of synthesizing the rhodamine NHS esters include (1) the
reaction condition of having substantially stoichiometric amounts of
di-N-succinimidylcarbonate (DSC) and 4-dimethylaminopyridine (DMAP)
present for esterification of the 5- or 6- forms of the rhodamine dyes to
produce high yields of product at room temperature, and (2) the treatment
of the freshly synthesized product with an acidic compound, preferably
having a pK.sub.a or less than 5, to prevent conversion back into
reactants. The general reaction scheme of the invention is defined by
Formula IX:
##STR3##
FORMULA IX
The methods comprise reacting the acid form of a 5- or 6-carboxylrhodamine
(either as a mixture of isomers, or as pure isomers) with equivalent
amounts of DSC and DMAP in a polar aprotic solvent to form the carboxyl
N-hydroxysuccinimide ester. Suitable polar aprotic solvents include
N,N-dimethylformamide (DMF), pyridine, hexamethylphosphoramide (HMPA), or
the like. Most preferably, DMF is used as the reaction solvent. The
isomerically mixed NHS esters can be separated into their individual
isomers for further use. Most preferably, in order to conserve reagents,
the acid forms of the 5- or 6-carboxylrhodamines are first separated into
their individual isomers by standard separative techniques, e.g. Edmundson
et al., Molecular Immunology, Vol. 21, pg. 561 (1984), and then the
individual 5- or 6- carboxyl isomers are reacted as described above to
form the 5- or 6-carboxyl NHS esters, respectively, which are separated
from the reaction mixture, again using standard techniques.
Preferably, the freshly synthesized rhodamine NHS ester is treated with a
volatile, organic-soluble acid with pK<5; and more preferably, a volatile,
organic-soluble acidic compound with pK<1, such as HCl or HBr in methanol,
or most preferably, trifluoroacetic acid.
Some isomeric mixtures of rhodamine dyes for use with the invention are
available commercially, e.g. Molecular Probes, Inc. (Eugene, Ore.), and
others can be synthesized in accordance with the teachings of U.S. Pat.
Nos. 2,242,572; 2,153,059; 3,822,270; 3,932,415; and 4,005,092, all of
which are incorporated by reference.
The rhodamines of the invention are particularly well suited for
identifying classes of polynucleotides that have been subjected to a
biochemical separation procedure, such as gel electrophoresis, where a
series of closely spaced bands or spots of polynucleotides having similar
physiochemical properties, e.g. size, conformation, charge,
hydrophobicity, or the like, are present in a linear or planar
arrangement. As used herein, the term "bands" includes any spacial
grouping or aggregation of polynucleotide on the basis of similar or
identical physiochemical properties. Usually bands arise in the separation
of dye-polynucleotide conjugates by gel electrophoresis.
Preferably, classes of polynucleotides identified in accordance with the
invention are defined in terms of terminal nucleotides so that a
correspondence is established between the four possible terminal bases and
the four spectrally resolvable dyes. In particular, classes of
polynucleotides arise in the context of the chain termination methods of
DNA sequencing, where dye-polynucleotide conjugates are separated
according to size by gel electrophoresis, e.g. Gould and Matthews, cited
above; Rickwood and Hames, Eds., Gel Electrophoresis of Nucleic Acids: A
Practical Approach, (IRL Press Limited, London, 1981); or Osterman,
Methods of Protein and Nucleic Acid Research, Vol. 1 (Springer-Verlag,
Berlin, 1984). Preferably the type of gel is polyacrylamide having a
concentration (weight to volume) of between about 2-20 percent. More
preferably, the polyacrylamide gel concentration is between about 4-8
percent. Preferably the gel includes a strand separating, or denaturing,
agent. Detailed procedures for constructing such gels are given by
Maniatis et al., "Fractionation of Low Molecular Weight DNA and RNA in
Polyacrylamide Gels Containing 98% Formamide or 7M Urea, " in Methods in
Enzymology, Vol. 65, pgs. 299-305 (1980); Maniatis et al., "Chain Length
Determination of Small Double- and Single-Stranded DNA Molecules by
Polyacrylamide Gel Electrophoresis," Biochemistry, Vol. 14, pgs.
3787-3794, (1975); and Maniatis et al., Molecular Cloning: A Laboratory
Manual (Cold Spring Harbor Laboratory, New York 1982), pgs. 179-185.
Accordingly these references are incorporated by reference. The optimal
gel concentration, pH, temperature, concentration of denaturing agent,
etc. employed in a particular separation depends on many factors,
including the size range of the nucleic acids to be separated, their base
compositions, whether they are single stranded or double stranded, and the
nature of the classes for which information is sought by electrophoresis.
Accordingly application of the invention may require standard preliminary
testing to optimize conditions for particular separations. Preferably,
during polynucleotide chain extension deoxyinosine triphosphate is
substituted for deoxyguanosine triphosphate to avoid so called "band
compression" during electrophoresis, e.g. Mills et al, Proc. Natl. Acad.
Sci., Vol. 76, pgs. 2232-2235 (1979). By way of example, polynucleotides
having sizes in the range of between about 10-500 bases are separated and
detected in accordance with the invention in the following gel: 6 percent
polyacrylamide made from 19 parts to 1 part acrylamide to bis-acrylamide,
formed in a Tris-borate EDTA buffer at pH 8.1 (measured at 25.degree. C.)
with 48 percent (weight/volume) urea. The gel is run at about 40.degree.
C.
The dye-polynucleotide conjugates on the gel are illuminated by standard
means, e.g. high intensity mercury vapor lamps, lasers, or the like.
Preferably, the dye-polynucleotides on the gel are illuminated by laser
light generated by a argon ion laser, particularly the 488 and 514 nm
emission lines of an argon ion laser. Several argon ion lasers are
available commercially which lase simultaneously at these lines, e.g.
Cyonics, Ltd. (Sunnyvale, Calif.) Model 2001, or the like.
An important feature of the invention is the DNA polymerase used for chain
extension in the DNA sequencing procedure. Preferably, the polymerase used
in the method of the invention is selected from the group consisting of
Taq DNA polymerase, described by Innis et al, Proc. Natl. Acad. Sci., Vol.
85, pgs. 9436-9440 (1988), with either a manganese or magnesium buffer;
and T7 DNA polymerase with a manganese buffer, described by Tabor et al,
Proc. Natl. Acad. Sci., Vol. 86, pgs. 4076-4080 (1989). Generally, the
single stranded DNA template containing the unknown sequence is prepared
by standard techniques, e.g. in M13mp18 as described in the manual for the
Applied Biosystems Model 370A DNA sequencing system. Likewise, the
annealing reaction, wherein the primer is attached to the template, is
carried out by standard protocols. The extension reaction, wherein
different-sized DNA fragments are generated is optimized for the
rhodamine-labeled dideoxynucleotides of the invention and the particular
DNA polymerase used.
EXAMPLES
The following examples serve to illustrate the present invention. The
concentrations of reagents, temperatures, and the values of other variable
parameters are only to exemplify the invention and are not to be
considered limitations thereof.
EXAMPLE 1
6-TMR-NHS
6-TMR acid was separated from a mixture of the 5- and 6-TMR acid isomers by
column chromatography. 8.82 mg of 6-TMR acid and 10.5 mg of DSC were
dissolved in 0.5 ml of dry DMF under argon. 0.09 ml of a 0.5 molar
solution of DMAP in tetrahydrofuran (THF) was added in one portion. After
2 hours at room temperature, the mixture was taken into 50 ml of
chloroform and washed three times with a 1:1 solution of brine:water. The
chloroform was evaporated and the residue was purified on a 20 g silica
gel column (300:30:8 methylene chloride:methanol:acetic acid elution).
Fractions with R.sub.f of about 0.4 were evaporated to dryness, yielding
8.6 mg of 6-TMR-NHS as its acetic acid salt.
EXAMPLE 2
5-TMR-NHS
5-TMR-NHS was prepared from 82.3 mg of 5-TMR acid, 75 mg of DSC, 0.70 ml of
0.5 molar DMAP in THF in 2 ml dry DMF, as described in Example 1.
EXAMPLE 3
6-ROX-NHS
6-ROX acid was separated from a mixture of 5- and 6- acid isomers by column
chromatography. 46.2 mg of 6-ROX acid and 58 mg of DSC were dissolved in 2
ml of dry DMF under argon and 0.45 ml of a 0.5 molar solution of DMAP in
THF was added in one portion. After 1.5 hours at room temperature, the
mixture was taken into 100 ml chloroform and washed four times with a 1:1
solution of brine:water. The chloroform was evaporated and the residue was
purified on a 40 g silica gel column (300:30:8 methylene
chloride:methanol:acetic acid elution). Fractions with R.sub.f of about
0.5 were evaporated to dryness, yielding 56.4 mg of 6-ROX-NHS as its
acetic acid salt.
EXAMPLE 4
5-ROX-NHS
5-ROX-NHS was prepared from 27.4 mg of 5-ROX acid, 30.2 mg of DSC, 0.24 ml
of 0.5 molar DMAP in THF in 1.0 ml dry DMF, as described in Example 3.
EXAMPLE 5
A Stable Formulation of Rhodamine NHS esters
a) 0.44 mg of 6-carboxy-X-rhodamine NHS ester from Example 3 and 80 ul of
0.01 molar ethanol amine in methanol were combined. Reverse phase HPLC of
the reaction mixture with acetonitrile and 0.1 molar triethylammonium
acetate buffer (pH=7.0) showed that the product was composed of 70%
X-rhodamine acid and 30% of X-rhodamine NHS ester (observed as the
ethanolamide of 6-carboxy-X-rhodamine from its reaction with ethanol
amine).
b) 0.15 g of 6-carboxy-X-rhodamine NHS ester from Example 3 were dissolved
in 100 m. of chloroform; the chloroform solution was washed two times with
0.5 molar sodium bicarbonate, dried with sodium sulfate, filtered, treated
with 0.1 ml of acetic acid and evaporated to dryness. 0.35 mg of the
product was treated exactly as in a); reverse phase HPLC showed 20%
6-carboxy-X-rhodamine acid and 80% of 6-carboxy-X-rhodamine NHS ester.
c) 0.15 g of 6-carboxy-X-rhodamine NHS ester from Example 3 was treated
exactly as in b), except that trifluoroacetic acid was substituted for
acetic acid. 0.19 mg of the resulting solid were treated exactly as in a);
reverse phase HPLC showed <5% 6-carboxy-X-rhodamine acid and >95%
6-carboxy-X-rhodamine NHS ester.
EXAMPLE 6
Preparation of R6G-labeled 7-deaza-2',3'-dideoxyadenosine triphosphate
(ddA-5-R6G)
To 2.0 umoles of 7-(3"-amino-1"-propynyl)-7-deaza-2',3'-dideoxyadenosine
triphosphate (lyophilized), obtained as described, is added 100 ul of DMF,
3 mg of 5-rhodamine 6G-NHS ester and 50 ul of 1.0M triethylammonium
carbonate, pH 8.95. This was vortexed and allowed to stand at room
temperature overnight. The mixture was then purified by HPLC on a AX-300
220.times.4.6 mm, 7 micron column with 1.5 ml per minute flow rate.
Starting elution was at 60% 0.1M triethylammonium carbonate, pH 7.0, 40%
CH.sub.3 CN with a linear gradient to 60% 1.2M triethylammonium carbonate,
pH 7.5, 40% CH.sub.3 CN over 40 minutes. The solvent was removed from the
collected product by evaporation under vacuum. The residue was dissolved
in 0.01M triethylammonium acetate pH 7.0 and quantified.
EXAMPLE 7
Preparation of ROX-labeled 2',3'-dideoxycytidine triphosphate (ddC-6ROX)
To 3.6 umoles of 5-(3"-amino-1"-propynyl)-2',3'-dideoxycytidine
triphosphate, (obtained as described) in 150 ul of H.sub.2 O was added 5
mg of 6-rhodamine X-NHS ester in 60 ul of DMSO and 50 ul of 1.0M
triethylammonium carbonate pH 8.95. This was vortexed and allowed to stand
at room temperature overnight. The product was purified as in Example 6.
EXAMPLE 8
Preparation of R110-labeled 2',3'-dideoxyinosine triphosphate (ddG-5R110)
To 1.3 umoles of 7-(3"-amino-1"-propynyl)-7-deaza-2',3'-dideoxyguanosine
triphosphate (lyophilized), obtained as described, was added 100 ul of
DMF, 4 mg of 5-rhodamine 110-NHS ester, and 100 ul of 1.0M
triethylammonium carbonate pH 8.95. This was vortexed and allowed to stand
overnight at room temperature. The product was purified as in Example 6.
EXAMPLE 9
Preparation of TMR-labeled 2',3'-dideoxythymidine triphosphate (ddT-6TMR)
3.1 umoles of 5-(3"-amino-1"-propynyl)-2',3'-dideoxyuridine triphosphate in
150 ul of H.sub.2 O, obtained as described, was mixed with 150 ul of DMF,
100 ul of 1.0M triethylammonium carbonate pH 8.95, and 4 mg of 6-TMR-NHS
ester. This was vortexed and allowed to stand overnight at room
temperature. The produce was purified as in Example 6.
EXAMPLE 10
DNA sequence analysis using rhodamine-labeled dideoxynucleotides and Taq
DNA polymerase with a magnesium buffer
The rhodamine labeled dideoxynucleotides prepared in Examples 6-9 were used
to label DNA fragments in chain termination sequencing using an Applied
Biosystems (Foster City, Calif.) Model 370A automated DNA sequencer. The
manufacturer's protocol (User Bulletin DNA Sequencer Model 370, Issue No.
2, Aug. 12, 1987), which is incorporated by reference, was followed for
obtaining M13mp18 single stranded template. (The M13 itself served as a
test sequence). The M13 universal primer was employed. The following
solutions were prepared: 5X Taq Mg Buffer (50 mM Tris-Cl pH 8.5, 50 mM
MgCl.sub.2, 250 mM NaCl); Dye-Terminator Mix (2.7 uM ddG-5R110, 9.00 uM
ddA-5R6G, 216.0 uM ddT-6TMR, and 54.0 uM ddC-6ROX); and DNTP Mix (500 uM
dITP, 100 uM dATP, 100 uM dTTP, and 100 uM dCTP). The annealing reaction
was carried out by combining in a microcentrifuge tube 3.6 ul of 5X Taq Mg
Buffer, 0.4 pmol DNA template, 0.8 pmol primer, and water to a volume of
12.0 ul. The mixture was incubated at 55.degree.-65.degree. C. for 5-10
minutes, cooled slowly over a 20-30 minute period to a temperature between
4.degree.-20.degree. C., then centrifuged once to collect condensation,
mixed, and placed on ice. To the mixture was then added 1.0 ul dNTP Mix,
2.0 ul Dye-Terminator Mix, 4 units of Taq polymerase, and water to bring
the volume to 18.0 ul. The mixture was incubated for 30 minutes at
60.degree. C., then placed on ice and combined with 25.0 ul of 10 mM EDTA
pH 8.0 to quench the reaction. The DNA in the mixture was then purified in
a spin column (e.g. a 1 ml Sephadex G-50 column, such as a Select-D from 5
Prime to 3 Prime, West Chester, Pa.) and ethanol precipitated (by adding 4
ul 3M sodium acetate pH 5.2 and 120 ul 95% ethanol, incubating on ice for
10 minutes, centrifuging for 15 minutes, decanting and draining the
supernatant, resuspending in 70% ethanol, vortexing, centrifuging for 15
minutes, decanting and draining the supernatant, and drying in a vacuum
centrifuge for 5 minutes). The precipitated DNA was then resuspended in 3
ul of a solution consisting of 5 parts deionized formamide and 1 part 50
mM EDTA pH 8.0 and vortexed thoroughly. Prior to loading on the gel the
mixture was incubated at 90.degree. C. for 2 minutes to denature the DNA.
Over 450 bases of the M13 plasmid were correctly identified by the base
calling routine of the Model 370A automated sequencer.
EXAMPLE 11
DNA sequence analysis using rhodamine-labeled dideoxynucleotides and T7 DNA
polymerase with a manganese buffer
The sequencing reaction is carried out as in Example 10, except that in
place of 5X Taq Mg Buffer 5X T7 Mn Buffer (100 mM Tris-Cl pH 7.5, 75 mM
sodium isocitrate, 10 mM MnCl.sub.2, 250 mM NaCl) is used, the
Dye-Terminator Mix consists of 1.8 uM ddG-5R110, 5.4 uM ddA-5R6G, 5.8 mM
ddt-6TMR, and 9.0 uM ddC-6ROX, the dNTP Mix consists of 750 uM dITP, 150
uM dATP, 150 uM dTTP, and 150 uM dCTP, and the extension reaction is
carried out at 37.degree. C. for 10 minutes.
EXAMPLE 12
DNA sequence analysis using rhodamine-labeled dideoxynucleotides and Taq
DNA polymerase with a manganese buffer
The sequencing was carried out as in Example 10, except in place of 5X Taq
Mg Buffer 5X Taq Mn Buffer (100 mM Tris-Cl pH 7.5, 75 mM sodium
isocitrate, 10 mM MnCl.sub.2, 250 mM sodium chloride) was used, and the
Dye-Terminator Mix consisted of 3.6 uM ddG-5R110, 2.7 uM ddA-5R6G, 43.2 uM
ddT-6TMR, and 28.8 uM ddC-6ROX. Over 450 bases of the M13 plasmid were
correctly identified by the base calling routine of the Model 370A
automated sequencer.
The foregoing disclosure of preferred embodiments of the invention has been
presented for purposes of illustration and description. It is not intended
to be exhaustive or to limit the invention to the precise form disclosed,
and obviously many modifications and variations are possible in light of
the above teaching. The embodiments were chosen and described in order to
best explain the principles of the invention and its practical
application, to thereby enable others skilled in the art to best utilize
the invention in various embodiments and with various modifications as are
suited to the particular use contemplated. It is intended that the scope
of the invention be defined by the claims appended hereto.
* * * * *
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