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Description  |
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BACKGROUND
The invention relates generally to molecular separation techniques, and
particularly to techniques for identifying oligonucleotides separated by
gel electrophoresis.
Many procedures in molecular biology require that heterogeneous mixtures of
DNA or RNA be electrophoretically separated into homogeneous components
according to mass, charge, conformation, isoelectric point, or the like.
The homogeneous components are then detected by densitometry or by
radioactive, fluorescent, or chromogenic labeling. Each such method of
identification has its own advantages and disadvantages, e.g. Gould and
Matthews, Separation Methods for Nucleic Acids and Oligonucleotides
(North-Holland Publishing Company, Amsterdam, 1976) pgs. 337-344. For
example, until recently DNA sequencing techniques relied exclusively on
radioactive labels for distinguishing oligonucleotides separated by
electrophoresis. Radioactive labels are highly sensitive, and can be
readily incorporated into the molecules of interest. However, there are
several inherent disadvantages to their use: In autoradiography resolution
is limited by the omnidirectional nature of the tracks of the decay
particles, the thickness and distance of the autoradiographic emulsion,
and the cumulative nature of the signal recorded in the emulsion.
Radioactive labels pose a laboratory health hazard, which requires that
the labels receive special handling and disposal. And, finally,
radioactive labels require long exposure or counting times for adequate
signal to noise resolution. This latter disadvantage is especially acute
when labels are used in conjunction with automated techniques, such as
automated DNA sequencing where bands of different kinds of labeled
nucleotides must be rapidly identified as they traverse a single
electrophoresis lane. Not only are there no nucleotide-specific
radioactive labels for practical identification, but even if there were,
current detection techniques such as autoradiography or scintillation
counting are too time consuming. As a consequence, fluorescent labeling
means have been sought for use with DNA sequencing techniques.
Fluorescent labels can be detected immediately after application; they are
conveniently handled; and they permit the precise localization and
quantification of the labeled molecules.
Several factors constrain the selection of fluorescent labels for an
oligomeric series undergoing separation by gel electrophoresis, such as an
oligomeric series of nucleotides whose members differ only in base number.
First, the labels must not adversely affect electrophoretic mobility so
that extensive band broadening occurs. Nor can the relative effects of the
labels on electrophoretic mobility be such that one or more band positions
become reversed or overlapping thereby destroying the correspondence
between band ordering and the natural order of the oligomeric series.
Unfortunately there is no reliable way to predict with certainty the
electrophoretic behavior of an oligomer with an arbitrarily chosen label
attached, such as an organic dye. Procedures for electrophoretic
separations are usually arrived at empirically; however, two major factors
determining electrophoretic mobility are charge and molecular weight.
Other important factors include configuration of the oligomers and gel
polymer density, Gould and Matthews, Separation Methods for Nucleic Acids
and Oligonucleotides (North-Holland Publishing Company, Amsterdam, 1976),
p. 313.
Second, where several distinct labels are required, a selection of dyes
cannot have significantly overlapping emission bands. However, given that
emission band halfwidth for organic fluorescent dyes is typically about
40-80 nanometers and that the width of the visible spectrum is only about
350-400 nanometers, it is exceedingly difficult to find a suitable
selection of fluorescent dyes without significant overlap whenever three
or more distinct fluorescent labels are required. Moreover, when several
fluorescent dyes are used, 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
together 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. Finally, the fluorescent labels must be
compatible with the chemistry used to create or manipulate the molecules
which are labeled. For example, in enzymatic sequencing of DNA, the
fluorescent dyes used to label primers cannot interfere with DNA
polymerase activity.
Smith et al, in "Synthesis of Oligonucleotides Containing an Aliphatic
Amino Group at the 5' Terminus: Synthesis of Fluorescent DNA Primers for
Use in DNA Sequence Analysis," Nucleic Acids Research, Vol. 13, pgs.
2399-2412 (1985), disclose a set of four fluorescent dyes for use in
enzymatic DNA sequence analysis for labeling oligonucleotides separated by
electrophoresis. Each dye from the set is used to identify on an
electrophoresis gel bands of oligonucleotides having the same 3' terminal
nucleotide.
SUMMARY OF THE INVENTION
In accordance with the method of the invention four sets of fluorescent
dyes are used to detect oligonucleotides whenever mixtures of up to four
classes of oligonucleotides are separated electrophoretically on a gel.
Members from each of the following sets of dyes have been found to be
spectrally resolvable with respect to members of every other set under the
gel electrophoretic conditions described below.
Set I consists of fluorescein derivatives defined by the formula:
##STR1##
wherein A is a linking functionality at the 5 or 6 carbon position which
can be used to link the fluorescein moiety of the dye to a complementary
functionality on an oligonucleotide,, and B is an acidic anionic group,
preferably carboxyl or sulfonic acid, and most preferably carboxyl.
The following table indicates illustrative linking functionalities
represented by A, their complementary functionalities, and the resulting
linking groups suitable for use with the invention.
______________________________________
Comple-
mentary
Linking Function- Linking
Functionality ality Group
______________________________________
NCS NH.sub.2 NHCSNH
##STR2## NH.sub.2
##STR3##
SO.sub.2 X NH.sub.2 SO.sub.2 NH
##STR4## NH.sub.2
##STR5##
##STR6## NH.sub.2
##STR7##
##STR8## SH
##STR9##
##STR10## SH
##STR11##
______________________________________
Preferably the linking functionality is isothiocyanate, sulfonyl chloride,
4,6-dichlorotriazinylamine, or succinimidyl carboxylate whenever the
complementary functionality is amine. And preferably the linking
functionality is maleimide, or iodoacetamide whenever the complementary
functionality is sulfhydryl.
Set II consists of derivatives of dichlorodimethoxyfluorescein defined by
the formula:
##STR12##
wherein A and B are defined as above.
Set III consists of tetramethylrhodamine derivatized with a linking
functionality at the 5 or 6 carbon position, as defined by the formula:
##STR13##
wherein A and B are defined as above.
Set IV consists of rhodamine X derivatives defined by the formula:
##STR14##
wherein A' is a linking functionality as represented by A (as defined
above) or an acidic anionic group as represented by B (as defined above),
and B' is an acidic anionic group whenever A' is a linking functionality
and B' is a linking functionality whenever A' is an acidic anionic group.
More preferably, A' is sulfonic acid or a linking functionality as
represented by A, and B' is carboxyl or sulfonic acid whenever A' is a
linking functionality, and B' is a linking functionality whenever A' is
sulfonic acid. In accordance with the invention, prior to separation,
members within each class of oligonucleotides are labeled with a dye
selected from the same set to form dye-oligonucleotide conjugates, such
that the members of different classes are labeled with dyes from different
sets. That is, each class corresponds to a different one of the sets I,
II, III, or IV defined above (also referred to herein as the first through
fourth sets, respectively). After labeling, the members of all classes are
combined to form a mixture. The mixture is then subjected to gel
electrophoresis in order to separate the oligonucleotides according to
mass, charge, conformation, and/or properties which form the bases of one
or two dimensional electrophoretic separations. Oligomeric series with
respect to such properties within and among the classes are determined by
the relative positions of similarly separated, e.g. bands, of
oligonucleotides on the gel. Finally, the dyes attached to the similarly
separated oligonucleotides are caused to fluoresce, and the identity of
their class is determined by the fluorescence or absorption spectrum of
the attached dye.
Class of oligonucleotides can arise in a variety of contexts. For example,
they can arise as products of restriction enzyme digests. Preferably,
classes identified in accordance with the invention are defined in terms
of the terminal nucleotides of nucleic acids so that a correspondence is
established between the four possible terminal bases and the four sets of
spectrally resolvable dyes. More preferably, the classes arise in the
context of chemical or enzymatic sequencing of nucleic acids, and most
preferably the classes arise in the context of enzymatic sequencing of
DNA. Necessary conditions for a class to be identifiable in accordance
with the invention are (1) that the oligonucleotides of the class be
capable of separation by gel electrophoresis, (2) that they be capable of
labeling by the dyes of the invention, and (3) that the classes be
mutually exclusive in that an oligonucleotide can only be a member of one
class.
As used herein the term "spectrally resolvable" means that the fluorescent
emission bands of the dyes within a set are sufficiently distinct, i.e.
sufficiently non-overlapping, from those of the dyes of every other set
such that the classes of oligonucleotide to which the dyes are attached
can be distinguished by standard photodetection systems.
Oligonucleotide as used herein means a single stranded or double stranded
chain of DNA or RNA in the size range of about 10-1000 bases in length (if
single stranded), or in the size range of about 10-1000 base pairs in
length (if double stranded).
The advantage of these sets of dyes arise from the nature of their spectral
properties in gel environments. In particular, the gel environments
suitable for electrophoretic separations cause a shift of about 10-15 nm
toward the red in the absorption and emission bands of the dyes of sets I
and II. Shifting of the absorption bands significantly increases the
efficiency with which the dyes can be excited with 514 nm light, a major
emission line of the argon ion laser, the most cost effective excitation
source. Also, the emission bands of dyes from set I are shifted away from
the 514 nm emission line significantly reducing the amount of scattered
light collected with the fluorescent signal from these dyes whenever the
dyes are illuminated with 514 nm light.
The method of the invention finds direct application to chemical and
enzymatic DNA sequencing techniques for fluorescently labeling
oligonucleotides separated by gel electrophoresis.
DETAILED DESCRIPTION OF THE INVENTION
The invention includes methods for detecting up to four predefined classes
of oligonucleotides that are electrophoretically separated according to
mass, charge, conformation, or other property on the same gel. The method
is accomplished by labeling oligonucleotides of each class with dyes
selected from a separate one of the four sets of dyes defined above. Such
labeling ensures that each class has a distinct and spectrally resolvable
fluorescent label.
Set I consists of fluorescein mono-derivatized with a linking functionality
at either the 5 or 6 carbon position (as determined by the Color Index
numbering system). Illustrative examples of set I members include
fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate (the -5- and
-6-forms being referred to collectively as FITC),
fluorescein-5-succinimidylcarboxylate,
fluorescein-6-succinimidylcarboxylate, fluorescein-5-iodoacetamide,
fluorescein-6-iodoacetamide, fluorescein-5-maleimide, and
fluorescein-6-maleimide. These examples of members of set I are available
commercially, e.g. Molecular Probes, Inc. (Junction City, OR), or can be
synthesized using standard techniques.
Set II consists of 2',7'-dimethoxy-4',5'-dichlorofluorescein
mono-derivatized with a linking functionality at the 5 or 6 carbon
position (the carbons being identified in accordance with the Color Index
numbering system). Set II members can be obtained by standard
modifications of
2,7-dimethoxy-4,5-dichloro-9-(2',4'-dicarboxyphenyl)-6-hydroxy-3H-xanthen-
3-one and
2,7-dimethoxy-4,5-dichloro-9-(2',5'-dicarboxyphenyl)-6-hydroxy-3H-xanthen-
3-one (IUPAC notation) disclosed in U.S. Pat. No. 4,318,846. Accordingly
U.S. Pat. No.4,318,846 is incorporated by reference. For example, the 4'
and 5' carboxys of these compounds can be condensed with
N-hydroxysuccinimide using dicyclohexylcarbodiimide to form an
amine-selective linking functionality, e.g. as illustrated by examples 6
and 8 of the above-referenced patent (Col. 24-29). Kasai et al., Anal.
Chem., Vol. 47, pages 34-37 (1975), discloses the basic technique for such
condensations. Accordingly Kasai et al. is incorporated by reference. Set
II dyes resulting from such reactions are
2',7'-dimethoxy-4',5'-dichlorofluorescein-5-succinimidylcarboxylate and
2,',7'-dimethoxy-4',5'-dichlorofluoescein-6-succinimidylcarboxylate (the
-5- and -6-forms being referred to collectively as DDFCS).
Set III consists of tetramethylrhodamine mono-derivatized with a linking
functionality at either the 5 or 6 carbon position. Illustrative examples
of set III members include tetramethylrhodamine-5-isothiocyanate,
tetramethylrhodamine-6-isothiocyanate (the -5- and -6-forms being referred
to collectively as TMRITC), tetramethylrhodamine-5-iodoacetamide,
tetramethylrhodamine-6-iodoacetamide,
tetramethylrhodamine-5-succinimidylcarboxylate,
tetramethylrhodamine-6-succinimidylcarboxylate,
tetramethylrhodamine-5-maleimide, and tetramethylrhodamine-6-maleimide.
These exemplary dyes are available commercially, e.g. Molecular Probes,
Inc., or can be synthesized using standard techniques.
Set IV consists of rhodamine X derivatives having a disubstituted phenyl
attached to the molecule's oxygen heterocycle, one of the substituents
being a linking functionality attached to the 4' or 5' carbon (IUPAC
numbering) of the phenyl, and the other being a acidic anionic group
attached to the 2' carbon. Illustrative examples of set IV members include
Texas Red (tradename of Molecular Probes, Inc.), rhodamine
X-5-isothiocyanate, rhodamine X-6-isothiocyanate, rhodamine
X-5-iodoacetamide, rhodamine X-6-iodoacetamide, rhodamine
X-5-succinimidylcarboxylate, rhodamine X-6-succinimidylcarboxylate,
rhodamine X-5-maleimide, and rhodamine X-6-maleimide. Most of these
exemplary dyes are available commercially, e.g. Molecular Probes, Inc., or
can be synthesized using standard techniques. For example, in the case of
Texas Red it can be synthesized according to the procedure disclosed in
Titus et al., "Texas Red, a Hydrophilic, Red-Emitting Fluorophore for Use
with Fluorescein in Dual Parameter Flow Microfluorometric and Fluorescence
Microscopic Studies," J. Immunological. Methods, Vol. 50, pgs. 193-204
(1982). 5- and 6-carboxy derivatives of rhodamine X can be synthesized
using standard techniques, e.g. as disclosed in U.S. Pat. No. 3,932,415,
which is incorporated by reference. The 5- or 6-carboxyl groups can then
be converted into linking functionalities by standard techniques. For
example, rhodamine X-succinimidylcarboxylate is formed by techniques
disclosed in Muller et al., Experimental Cell Research, Vol. 100, pgs.
213-217 (1976). Accordingly, this reference is incorporated by reference.
The dyes are attached to oligonucleotides using standard procedures, e.g.
for a review see Haugland, "Covalent Fluorescent Probes," in Excited
States of Biopolymers, Steiner, Ed. (Plenum Press, New York, 1983), pgs.
29-58, which pages are incorporated by reference. Recently several
techniques have been developed for attaching reactive functionalities to
oligonucleotides making it possible to form covalent dye-oligonucleotide
conjugates by condensing the reactive functionality on the oligonucleotide
with a linking functionality of a dye. For example, Smith et al., cited
above, discloses a procedure for attaching an amine group to the 5' end of
an oligonucleotide, and Connolly and Rider, "Chemical Synthesis of
Oligonucleotides Containing a Free Sulphydryl Group and Subsequent
Attachment of Thiol Specific Probes," Nucleic Acids Research, Vol. 13,
pgs. 4485-4502 (1985), discloses a procedure for attaching sulphydryl
groups. Accordingly these two references are incorporated by reference.
Preferably, the reactive, or complementary, functionality on the
oligonucleotides is an amine. And preferably the reactive amine is
attached by way of the linking agents disclosed in copending U.S. patent
application Ser. No. 769,170 filed Aug. 26, 1985, entitled
"Amino-derivatized Phosphite and Phosphate Linking Agents, Phosphoramidite
Precursors, and Useful Conjugates Thereof." Accordingly this application
is incorporated by reference. Most preferably the reactive amine is
attached by reacting
2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane with the
oligonucleotides. Standard electrophoretic procedures are employed for
separating the labeled nucleic acids, 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 nucleic acids separated are
oligonucleotides.
Preferably the type of gel is polyacrylamide having a concentration (weight
of 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 Ggel
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. By way of example, oligonucleotides
having sizes in the range of between about 20-300 bases have been
separated and detected in accordance with the invention in the following
gel: 5 percent polyacrylamide made from 25 parts to 1 part acrylamide to
bis-acrylamide, formed in a Tris-borate EDTA buffer at pH 8.3 (measured at
25.degree. C.) with 48 percent (weight/volume) urea. The gel was run at
50.degree. C.
The dye-oligonucleotide conjugates on the gel are illuminated by standard
means, e.g. high intensity mercury vapor lamps, lasers, or the like.
Preferably, the dye-oligonucleotides 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, CA) Model 2001, or the like.
I. SYNTHESIS OF A PREFERRED LINKING AGENT:
2-METHOXY-3-TRIFLUOROACETYL-1,3,2-OXAZAPHOSPHACYCLOPENTANE
Chloro-N,N-diisopropylaminomethoxy phosphine (5.0 ml, available form
Aldrich Chemical Co., Milwaukee, WI) was added dropwise at 0.degree. C. to
a stirred solution of N-(2-hydroxyethyl)-2,2,2-trifluoroacetamide (3.9 g)
and diisopropylethylamine (5.0 ml) in dichloromethane (about 40 ml) under
argon. (N-(2-hydroxyethyl)-2,2,2-trifluoroacetamide is synthesized
following the procedures disclosed by Lazarus and Benkovic in J. Am. Chem.
Soc., Vol. 101, pgs 4300-4312 (1979): Ethyl trifluoroacetate (56.8 g, 0.4
mol) in 50 mL of chloroform is added dropwise to a stirred solution of
24.4 (0.4 mol) of ethanolamine in 50 mL of chloroform. The solution is
stirred at room temperature for 5 h, rotary evaporated to remove the
solvent, and distilled at 115.degree. C. (4.3 Torr) to give 58.5 g of oil
that crystallized upon scratching.) After stirring at room temperature for
0.5 hours the reaction mixture was washed twice with 0.2M potassium
carbonate solution and once with brine, dried (MgSO.sub.4), and
concentrated under reduced pressure to give
N-(2-(N',N'-diisopropylaminomethoxyphosphinyloxy)ethyl)-2,2,2-trifluoroace
tamide as a colorless liquid (7.77 g).
.sup.1 H-NMR (CD.sub.2 Cl.sub.2): .delta.3.6 (6H, m), 3.4 (3H, d, J=12),
1.2 (12H, d, J=6.5)
.sup.31 P-NMR (CD.sub.2 Cl.sub.2, .sup.1 H decoupled): .delta.149(s)
N-(2-(N',N'-diisopropylaminomethoxyphosphinyloxy)ethyl)-2,2,2-trifluoroacet
amide (7.7 g) was distilled (58.degree.-59.degree. C. at 0.8 Torr) to
quantitatively yield
2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane as a colorless
liquid.
IR (film): 1705, 1420, 1230, 1200, 1160, 1020, 965 cm.sup.-1
.sup.1 H-NMR (CD.sub.2 Cl.sub.2): .delta.4.45 (2H, m), 3.65 (2H, m), 3.60
(3H, d, J=12)
.sup.31 P-NMR (CD.sub.2 Cl.sub.2, .sup.1 H decoupled): .delta.132(s), 125
(q, J=61)
MS: m/e 217 (M.sup.+), 197, 148, 136, 123, 120, 109, 92, 79, 70(100), 69,
62
II. REACTING 2-METHOXY-3-TRIFLUOROACETYL-1,3,2-OXAZAPHOSPHACYCLOPENTANE
WITH THE 5' TERMINUS OF AN OLIGONUCLEOTIDE TO FORM A
5'-(PROTECTED)-AMINOOLIGONUCLEOTIDE
Attachment of 2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane to
a 5' hydroxyl of an oligonucleotide was performed on an Applied Biosystems
380A DNA synthesizer (Applied Biosystems, Foster City, CA), or comparable
instrument. Caruthers et al, U.S. Pat. No. 4,458,066; Caruthers et al,
U.S. Pat. No. 4,415,732; and Caruthers et al, "New Methods for
Synthesizing Deoxyoligonucleotides," in Genetic Engineering, Vol. 4, pgs.
1-17 (Plenum Press, New York, 1982) provided detailed descriptions of the
chemistry used by the Applied Biosystems 380A DNA synthesizer.
Accordingly, these references are incorporated by reference for those
descriptions. 2-Methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane
was used as a 0.2M acetonitrile solution in combination with 0.5M
tetrazole/acetonitrile solution to form an activated reagent in the
synthesis cycle. The normal synthesizer cycle was modified only during the
addition of the activated reagent in the following manner. The activated
reagent was added twice with 1 hour wait times after each addition. The
coupling yields were about 95%. Normal deprotection with
thiophenol/triethylamine and then ammonium hydroxide gave a
5'-aminoethylphosphate oligonucleotide. Similar yields were obtained when
the activated reagent comprised an acetonitrile solution containing 0.2M
2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane and 0.1M
4-dimethylaminopyridine. In this case the modified activator reagent was
added once, and allowed to react for about 2-3 minutes.
III. ATTACHING DYES TO THE AMINO-DERIVATIZED OLIGONUCLEOTIDES
The trifluoroacetyl protection group is removed from the linking agent by
treatment with concentrated ammonium hydroxide to give
5'-aminoethylphosphate oligonucleotides. Attachment of the dyes to the
exposed amino groups is accomplished by standard procedures, such as the
ones described in the following examples.
A. FITC
A DMF solution of FITC (25 microliters at a concentration of 10 mg/ml, e.g.
available from Molecular Probes, Inc., Junction City, OR) is added to a
solution of 5'-aminoethylphosphate oligonucleotide (an 18-mer) (0.20
micromolar) in water (200 microliters) and 1M NaHCO.sub.3 /Na.sub.2
HCO.sub.3 buffer, pH 9.0 (25 microliters). The resulting solution is
stired in the dark for 6 hours or more. To remove the unconjugated dye,
the reaction is passed through an equilibrated 10 ml Sephadex G-25
(medium) (a trademarked product of Pharmacia Fine Chemicals) column with
water. The band of colored material eluting in the excluded volume is
collected. The crude 5'-fluorescein aminoethylphosphate oligonucleotide is
purified by polyacrylamide gel electrophoresis or by HPLC (e.g.
Perkin-Elmer Series 4, or comparable device) on a Vydac C18 column (No.
218TP54), or the like, in a linear gradient of 10-20% acetonitrile/0.1M
triethylammonium acetate, pH 7.0.
B. TMRITC
A DMF solution of TMRITC (10 microliters at a concentration of 20 mg/ml,
e.g. available from Research Organics, Inc., Cleveland, OH, or Molecular
Probes, Inc., Junction City, OR) is added to a solution of
5'-aminoethylphosphate oligonucleotide (an 18-mer) (0.004 micromole) in
water (88 microliters) and 1M NaHCO.sub.3 /Na.sub.2 CO.sub.3 buffer, pH
9.0 (2 microliters). The resulting solution is stored in the dark for 6
hours or more. The reaction is passed through an equilibrated 10 ml
Sephadex G-25 (medium) column with 0.1M triethylammonium acetate, pH 7.0.
The band of colored material in the excluded volume is purified as for
FITC.
C. Texas Red
The procedure for attaching Texas Red to the 5'-aminoethylphosphate
oligonucleotides can be accomplished by following the same procedure as
for TMRITC.
D. DDFCS
DDFCS (0.3 mg) was added to a solution of 5'-aminoethylphosphate
oligonucleotide (an 18-mer) (0.006 micromoles in 10 microliters of water)
and 1M NaHCO.sub.3 /Na.sub.2 CO.sub.3 buffer, pH 9.0 (10 microliters). The
resulting solution was stored in the dark for 5 hours and worked up as for
FITC.
IV. USE OF DYE-AMINOETHYLPHOSPHATE OLIGONUCLEOTIDE CONJUGATES AS PRIMERS IN
ENZYMATIC DNA SEQUENCE ANALYSIS
DNA sequence analysis is highly useful, both scientifically and
commercially. The two primary techniques for sequencing DNA fragments are
chemical methods, e.g., Maxam and Gilbert, Proc. Nat. Acad. Sci., Vol. 74,
p. 560 (1970), and enzymatic replication methods, e.g., Smith, Methods in
Enzymology, Vol. 65, Grossman and Moldave, eds., pgs. 560-580 (Academic
Press, New York, 1980), and Sanger et al., Proc. Natl. Acad. Sci., Vol.
74, pgs. 5363-5367 (1977). The method of the invention can be applied with
either technique to substitute fluorescent labels for radioactive labels.
In this example, it is shown how the subject invention is used in the
enzymatic DNA sequencing method of Sanger et al, "Cloning in
Single-Stranded Bacteriophage as an Aid to Rapid DNA Sequencing," J. Mol.
Biol., Vol. 143, pgs. 161-178 (1980), and Schreier and Cortese, "A Fast
Simple Method for Sequencing DNA Cloned in the Single-Stranded
Bacteriophage M13," J. Mol. Biol., Vol. 129, pgs. 169-172 (1979), both
references being incorporated herein by reference. The DNA sequencing
method described by these references will be referred to as the "Sanger
method." Before the Sanger method is described, it will be useful to
define the following terms.
DNA polymerase is a large multi-function enzyme which catalyzes the
synthesis of single-stranded DNA. The particular kind of DNA polymerase
used in the Sanger method is the so-called Klenow fragment of Escherichia
coli DNA polymerase I. This fragment possesses the synthetic function of
the enzyme. For synthesis DNA polymerase requires a template, a primer,
and a source of deoxyribonucleotides.
A template is a single-stranded piece of DNA which determines the sequence
of nucleotides in the single-stranded piece of DNA synthesized by the DNA
polymerase. During synthesis, the DNA polymerase moves along the template,
and for each nucleotide base thereof, the DNA polymerase attaches the
complementary nucleotide to the growing chain of single-stranded DNA. A
complementary nucleotide base is one associated with a given base in
accordance with the base-pairing rule for the formation of double-stranded
DNA. The base-pairing rule requires that adenosine of one strand always be
paired with thymidine of the other strand, and that cytidine of one strand
always be paired with guanosine of the other strand. Thus, when the DNA
polymerase encounters an adenosine on the template, it adds a thymidine to
the chain being synthesized, the when it encounters a cytidine, it adds a
guanosine. After the DNA polymerase moves on, the newly synthesized chain
and the complementary portion of the template are in double-stranded form.
A primer is a fragment of single-stranded DNA. The primer provides a
starting location for the DNA polymerase to begin adding nucleotides in
the synthesis process. The primer must be annealed to the piece of
single-stranded DNA containing the template so that a section of
double-stranded DNA is provided as the starting point for the DNA
polymerase.
Dideoxyribonucleotides are identical to deoxyribonucleotides except that
they lack both the 2' and 3' hydroxyl groups on the ribose moiety, instead
of just the 2' hydroxyl as with deoxyribonucleotides.
Dideoxyribonucleotides are sometimes referred to as analogs of
deoxyribonucleotides, in that DNA polymerase accepts the dideoxy
derivatives in place of the corresponding deoxyribonucleotide in the DNA
synthesis process. When such a substitution takes place, synthesis stops
because the DNA polymerase has no 3' hydroxy group on which to attach the
subsequent nucleotide.
In the Sanger method a DNA strand to be sequenced is used as a template for
Escherichia coli DNA polymerase I. A primer is annealed to a piece of
single-stranded DNA containing the template, and then it is extended
enzymatically to an average of 20 to 300 or more nucleotides in the
presence of radioactively labeled deoxyribonucleoside triphosphates, e.g.
.sup.32 P-labeled adenosine triphosphate, and the dideoxyribonucleoside
triphosphate analog of one of the four nucleotides. That is, four separate
reactions are carried out each including a different dideoxy analog.
Because DNA chain growth requires the addition of deoxyribonucleotides to
the 3'-hydroxyl, incorporation or a dideoxyribonucleotide terminates chain
growth. Incorporation of the dideoxy analog in place of the normal
nucleotide occurs randomly, so that each of the four reactions generates a
heterogeneous population of labeled strands terminating with the same
nucleotide, which can be separated electrophoretically according to chain
length. That is, four classes of oligonucleotides are established based on
the type of terminal dideoxyribonucleoside which is present. A single
stranded DNA phage M13 is used to clone copies of the DNA fragment to be
sequenced. When a sufficient quantity of M13 is cloned, the M13 DNA is
purified and separated into four aliquots. In each aliquot the synthesis
or chain growth reaction takes place in the presence of the respective
dideoxyribonucleotides.
In accordance with the invention, instead of labeling oligonucleotides by
incorporation of radioactive nucleotides during the chain growth phase,
primers are synthesized and then labeled by attaching a linking
functionality and reacting it with a dye. Preferably an amine linking
functionality is attached by reacting the primers with
2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane to form
5'-(protected)aminoethylphosphate oligonucleotides. The protecting groups
are removed and a dye of the invention is attached to the deprotected
5'-amine to form dye-primer conjugates. The dye-primer conjugates are then
used in accordance with Sanger's method, with the exception that
oligonucleotides from the four aliquots are mixed together and loaded onto
the same electrophoresis lane. The relative size of the oligonucleotides
and the nature of their terminal dideoxyribonucleotides are determined as
bands of homogeneous oligonucleotides travel down the electrophoresis lane
and are detected by a fluorimeter or spectrophotometer after illumination.
In accordance with the invention the bands are preferably illuminated with
both 514 nm and 488 nm laser limit, either sequentially or simultaneously.
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