U.S. patent number 5,707,804 [Application Number 08/410,808] was granted by the patent office on 1998-01-13 for primers labeled with energy transfer coupled dyes for dna sequencing.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Alexander Glazer, Jingyue Ju, Richard Mathies.
United States Patent |
5,707,804 |
Mathies , et al. |
January 13, 1998 |
Primers labeled with energy transfer coupled dyes for DNA
sequencing
Abstract
Compositions are provided for making oligonucleotides carrying
pairs of donor and acceptor dye molecules, designed for efficient
excitation of the donor at a single wavelength and emission from
the acceptor in each of the pairs at different wavelengths. The
different molecules having different donor-acceptor pairs can be
modified to have substantially the same mobility under separation
conditions, by varying the distance between the donor and acceptor
in a given pair, and to have enhanced emission intensities from the
acceptor. Particularly, the fluorescent compositions find use as
labels in sequencing nucleic acids.
Inventors: |
Mathies; Richard (El Cerrito,
CA), Glazer; Alexander (Orinda, CA), Ju; Jingyue
(Berkeley, CA) |
Assignee: |
The Regents of the University of
California (Oakland, CA)
|
Family
ID: |
22699330 |
Appl.
No.: |
08/410,808 |
Filed: |
March 27, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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189924 |
Feb 1, 1994 |
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Current U.S.
Class: |
435/6.12;
536/24.3 |
Current CPC
Class: |
C12Q
1/6818 (20130101); G01N 33/542 (20130101); G01N
33/582 (20130101); C12Q 1/6818 (20130101); C12Q
2537/143 (20130101); C12Q 2565/1015 (20130101); Y10S
435/81 (20130101) |
Current International
Class: |
G01N
33/536 (20060101); C12Q 1/68 (20060101); G01N
33/58 (20060101); G01N 33/542 (20060101); C12Q
001/68 (); C07H 021/04 () |
Field of
Search: |
;435/6
;536/23.1,24.3 |
References Cited
[Referenced By]
U.S. Patent Documents
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4855225 |
August 1989 |
Fung et al. |
4868103 |
September 1989 |
Stavrianopoulos et al. |
4996143 |
February 1991 |
Heller et al. |
5188934 |
February 1993 |
Menchen et al. |
5326692 |
July 1994 |
Brinkley et al. |
5342789 |
August 1994 |
Chick et al. |
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Foreign Patent Documents
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86116652.3 |
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Dec 1986 |
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EP |
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WO9214845 |
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Sep 1992 |
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WO |
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WO9309128 |
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May 1993 |
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WO |
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WO9417397 |
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Aug 1994 |
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WO |
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Other References
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Wei et al. (1994) Anal. Biochem. 66:1500-6. .
Ju et al., Fluorescence Energy Transfer Dye-Labeled Primers for DNA
Sequencing and Analysis, (1995) Proc. Natl. Acad. Sci. USA,
92:4347:4351. .
Smith et al., Fluorescence Detection in Automated DNA Sequence
Analysis, (1986) Nature, 321:674-679. .
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Chain-Terminating Dideoxynucleotides, (1987) Science, 238:336-341.
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Ansorge et al., Automated DNA Sequencing: Ultrasensitive Detection
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Research, vol. 15 No. 11:4593-4603. .
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Glazer et al., Fluorescent Tandem Phycobiliprotein Conjugates,
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In Solution By Fluorescence Resonance Energy Transfer, (1993) Proc.
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Acid Structures And Sequences, (1994) Nucleic Acids Research, vol.
22, No. 6:920-928. .
Selvin et al., Luminescence Energy Transfer Using A Terbium
Chelate: Improvements On Fluorescence Energy Transfer, (1994) Proc.
Natl. Acad. Sci, USA, 91:10024-10028. .
L. Stryer, Fluorescence Energy Transfer As A Spectroscopic
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6:1062-1070..
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Primary Examiner: Jones; W. Gary
Assistant Examiner: Atzel; Amy
Attorney, Agent or Firm: Townsend and Townsend and Crew
LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of application Ser. No.
08/189,924, filed Feb. 1, 1994.
Claims
What is claimed is:
1. A method of identification and detection of components in a
multicomponent mixture employing different fluorescent labels to
detect at least two components of interest, wherein said labels are
characterized by: (1) having a donor-acceptor fluorescent pair
bonded to an oligonucleotide chain with energy transfer from said
donor to said acceptor; and (2) each of the labels absorbs at
substantially the same wavelength and emits at a different
wavelength;
said method comprising:
binding different labels to different components of said
multi-component mixture;
detecting each of said labeled components by irradiating at the
absorption wavelength of said donor and detecting the fluorescence
of each of said labels.
2. A method according to claim 1, wherein said donor absorbs light
in the wavelength range of 350-800 nm and said acceptor emits light
in the wavelength range of 450-1000 nm.
3. A method according to claim 2, wherein said acceptor-donor pair
are 9-phenylxanthenes.
4. A method of separating components of a multicomponent mixture,
wherein each of the different components of interest are labeled
with different labels, wherein said labels are characterized by:
(1) having a donor-acceptor fluorescent pair bonded to an
oligonucleotide chain with efficient energy transfer from said
donor to said acceptor; (2) each of the labels absorbs at
substantially the same wavelength and emits at a different
wavelength; (3) and each of the different labels has substantially
the same mobility in said separation as a result of varying the
spacing of said donor-acceptor pair along said oligonucleotide
chain;
said method comprising:
binding different labels to different components of said
multi-component mixture;
separating said multi-component mixture into individual fractions;
and
detecting each of said labeled components by irradiating at an
absorption wavelength of said donor and detecting the fluorescence
of each of said labels.
5. A method according to claim 4, wherein said separation is by
electrophoresis.
6. A method according to claim 4, wherein said donor absorbs light
in the wavelength range of 350-800 nm and said acceptor emits light
in the wavelength range of 450-1000 nm.
7. A method according to claim 6, wherein said acceptor-donor pair
are 9-phenylxanthenes.
8. In a method for sequencing a nucleic acid which employs primers
for copying a single stranded nucleic acid and dideoxynucleotides
for terminating the chain at a particular nucleotide resulting from
said copying, said method comprising:
cloning a nucleic acid fragment to be sequenced into a vector
comprising a primer binding sequence 5' to said fragment
complementary to a primer, or ligating an oligonucleotide primer
binding sequence to a DNA fragment to be sequenced, where the
sequence of said oligonucleotide is complementary to a primer;
copying said fragment with a DNA polymerase in the presence of said
primer, dNTPs and each of a plurality of dideoxynucleotides in
separate reaction vessels, to generate single stranded DNA
sequencing fragments; and
separating the resulting mixture of single stranded DNA sequencing
fragments and determining the sequence by means of the bands
present on the gel;
the improvement which comprises:
employing primers which are characterized by:
(1) having an acceptor-donor fluorescent pair bonded to a nucleic
acid chain complementary to said primer binding sequence, where the
donor transfers energy to said acceptor for enhanced fluorescence
of said acceptor; (2) each of the primers absorbs at substantially
the same wavelength and emits at a different wavelength; and (3)
each of the primers has substantially the same mobility in said
separation, resulting from varying the spacing and fluorophores of
said donor-acceptor pair along said nucleic acid chain.
9. A method according to claim 8, wherein one of the members of
said acceptor-donor fluorescent pair is bonded to the 5' terminus
of said primer.
10. A method according to claim 8, wherein there are four primers
having different acceptor-donor pairs.
11. A method according to claim 8, wherein said acceptor-donor
fluorescent pair is separated by not more than 20 nucleotides.
12. A method according to claim 8, wherein at least two
acceptor-donor fluorescent pairs are xanthene compounds.
13. A method according to claim 12, wherein said xanthene compounds
comprise fluorescein derivatives and rhodamine derivatives.
Description
TECHNICAL FIELD
The field of this invention is fluorescent tags and their use for
DNA sequencing.
BACKGROUND
There is an increasing demand to be able to identify and quantify
components of mixtures. The greater the complexity of the mixture,
the greater the interest in being able to simultaneously detect a
plurality of the components present. As illustrative of this
situation is DNA sequencing, where it is desirable to efficiently
excite from one to four fluorescently tagged components with a
laser source at a single wavelength, while providing for
fluorescent signal emission at a plurality of distinctive
wavelengths. In this situation, the different labels should not
adversely affect the electrophoretic mobility of the sequences to
which they are attached.
Currently, there are four methods used for automated DNA
sequencing: (1) the DNA fragments are labeled with one fluorophore
and then the fragments run in adjacent sequencing lanes (Ansorge et
al., Nucleic Acids Res. 15, 4593-4602 (1987); (2) the DNA fragments
are labeled with four different fluorophores and all the fragments
are electrophoretically separated and detected in a single lane
(Smith et al., Nature 321, 674-679 (1986); (3) each of the
dideoxynucleosides in the termination reaction is labeled with a
different fluorophore and the four sets of fragments are run in the
same lane (Prober et al., Science 238, 336-341 (1987); or (4) the
sets of DNA fragments are labeled with two different fluorophores
and the DNA sequences coded with the dye ratios (Huang et al.,
Anal. Chem. 64, 2149-2154 (1992).
All of these techniques have significant deficiencies. Method 1 has
the potential problems of lane-to-lane variations in mobility, as
well as a low throughput. Methods 2, 3 and 4 require that the four
dyes be well excited by one laser source and that they have
distinctly different emission spectra. In practice, it is very
difficult to find two or more dyes that can be efficiently excited
with a single laser and that emit well separated fluorescent
signals.
As one selects dyes with distinctive red-shifted emission spectra,
their absorption maxima will also move to the red and all the dyes
can no longer be efficiently excited by the same laser source.
Also, as more different dyes are selected, it becomes more
difficult to select all the dyes such that they cause the same
mobility shift of the labeled molecules.
It is therefore of substantial interest that improved methods be
provided which allow for multiplexing of samples, so that a
plurality of components can be determined in the same system and in
a single run. It is also desirable for each label to have strong
absorption at a common wavelength, to have a high quantum yield for
fluorescence, to have a large Stokes shift of the emission, that
the various emissions be distinctive, and that the labels introduce
the same mobility shift. It is difficult to accomplish these
conflicting goals by simply labeling the molecules with a single
dye.
SUMMARY OF THE INVENTION
The subject invention provides compositions and methods for
analyzing a mixture using a plurality of fluorescent labels. To
generate the labels, pairs or families of fluorophores are bound to
a backbone, particularly a nucleic acid backbone, where one member
of each family is excited at approximately a common wavelength. By
exploiting the phenomenon of energy transfer, the other members of
each of the families emit at detectably different wavelengths. The
range of distances between donor and acceptor chromophores is
chosen to ensure efficient energy transfer. Furthermore, labels
used conjointly are selected to have approximately the same
mobility in a separation system. This is achieved by changing the
mobility of the labeled entity by varying the distance between the
two or more members of the family of fluorophores and choosing
labels with the same mobility. The subject invention finds
particular application in DNA sequencing, where the fluorophores
may be attached to universal or other primers and different
fluorophore combinations used for the different dideoxynucleosides.
Kits of combinations of labels are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
The file of this patent contains at least one drawing executed in
color. Copies of this patent with color drawings will be provided
by the Patent and Trademark Office upon request and payment of the
necessary fee.
FIG. 1. Structures of the twenty energy transfer (ET) primers and a
representative synthetic scheme for the preparation of F3T.
FIG. 2A, 2B, 2C and 2D shows the comparison of the fluorescence
emission intensity of the four energy transfer (ET) primers (F10F,
F10J, F3T and F3R) with the corresponding single dye-labeled
primers at the indicated excitation wavelength (1.times.TBE, 7M
urea). The thick lines indicate the absorption spectra of the ET
primers. FIG. 2A shows F10F vs. FAM, FIG. 2B shows F10J vs. JOE,
FIG. 2C shows F3T vs. TAMRA and FIG. 2D shows F3R vs. ROX. The
emission spectra for each primer pair were determined using
solution having the same molar concentration.
FIG. 3 shows the normalized fluorescence emission spectra of the
four ET primers (F10F, F10J, F3T and F3R) (1.times.TBE, 7M
urea).
FIG. 4A, 4B, 4C, and 4D show that the fluorescence emission
intensity of the ET primers is increased as the distance between
the donor and acceptor increases. The emission spectra for each
primer series were determined at the same molar concentration in
1.times.TBE.
FIG. 5 shows capillary electropherograms of each ET primer series.
A separate experiment has established that F10F, F10J, F3T and F3R
have very similar mobilities. The mobilities of the other primers
are shown for each set, relative to that of F10F, F10J, F3T and
F3R, respectively. Sample was analyzed by typical capillary
electrophoresis (CE) DNA sequencing conditions with 488 nm
excitation.
FIG. 6 shows that the mixed single base (ddATP/dNTPs) DNA
sequencing fragments generated with F10F, F10J, F10T and F10R
individually and then combined together have substantially the same
mobility shift. Samples were prepared using Sequenase 2.0 Kit
(USB/Amersham LIFE SCIENCE) and run on a 4-color CE DNA
sequencer.
FIG. 7 shows a portion of 4-color raw data (base 157 to 196) of DNA
sequencing profile of M13mp 18 DNA using the ET primer F10F, F10J,
F10T and F10R and Sequenase 2.0. Primer concentration: 0.4 pmol;
DNA template: 0.8 .mu.g (0.2 .mu.g for each base extension).
FIGS. 8A, 8B, 8C, 8D and 8E show the comparison of signal strengths
and mobility shifts of the single dye-labeled primers and ET
primers. A total of eight sequencing reactions with ddTTP/dNTPs
were run using 1 .mu.g of M13mp18 DNA template and 0.4 pmol of
primer and then loaded in 8 adjacent lanes of the ABI 373A
sequencing gel. FIG. 8A shows the raw traces obtained when single
dye-labeled primers were used. Colors correspond to the dye as
follows: blue, FAM; green, JOE; black, TAMRA; red, ROX. The region
shown corresponds to the sequence approximately 15-35 bases from
the 3' end of the primer. FIG. 8B shows raw traces on identical
scales obtained using ET primers. Colors correspond to the dye as
follows: blue, F10F; green, F10J; black, F3T; red, F3R. FIGS. 8C
and 8D display data from 4-color sequencing reactions run with
single-dye primers (C) and ET primers (D) on identical scales. For
reference, the ET primer data in (D) is also shown in analyzed
format in FIG. 8E. The reactions used for panel c included 0.4 pmol
of FAM and JOE primer; 0.8 pmol of TAMRA and ROX primer, and the
reactions for FIGS. 8D and 8E included 0.4 pmol of each ET primer
and a total of 6 .mu.g of M13mp18 template DNA.
DESCRIPTION OF SPECIFIC EMBODIMENTS
Novel fluorescent labels, combinations of fluorescent labels, and
their use in separation systems involving the separation of a
plurality of components are provided. Particularly, the fluorescent
labels comprise pairs of fluorophores, which with one exception
where the fluorophores are the same, involve different fluorophores
having overlapping spectra, where the donor emission overlaps the
acceptor absorption, so that there is energy transfer from the
excited fluorophore to the other member of the pair. It is not
essential that the excited fluorophore actually fluoresce, it being
sufficient that the excited fluorophore be able to efficiently
absorb the excitation energy and efficiently transfer it to the
emitting fluorophore.
The donor fluorophores in the different families of fluorophores
may be the same or different, but will be able to be excited
efficiently by a single light source of narrow bandwidth,
particularly a laser source. The door fluorophores will have
significant absorption, usually at least about 10%, preferably at
least about 20% of the absorption maxima within 20 nm of each
other, usually within 10 nm, more usually within 5 nm, of each
other. The emitting or accepting fluorophores will be selected to
be able to receive the energy from donor fluorophores and emit
light, which will be distinctive and detectably different.
Therefore, one will be able to distinguish between the components
of the mixture to which the different labels have been bound.
Usually the labels will emit at emission maxima separated by at
least 10 nm, preferably at least 15 nm, and more preferably at
least 20 nm.
Usually the donor fluorophores will absorb in the range of about
350-800 nm, more usually in the range of about 350-600 nm or
500-750 nm, while the acceptor fluorophores will emit light in the
range of about 450-1000 nm, usually in the range of about 450-800
nm. As will be discussed subsequently, one may have more than a
pair of absorbing molecules, so that one may have 3 or more
molecules, where energy is transferred from one molecule to the
next at higher wavelengths, to greatly increase the difference in
wavelength between absorption and observed emission.
The two fluorophores will be joined by a backbone or chain, usually
a polymeric chain, where the distance between the two fluorophores
may be varied. The physics behind the design of the labels is that
the transfer of the optical excitation from the donor to the
acceptor depends on 1/R.sup.6, where R is the distance between the
two fluorophores. Thus, the distance must be chosen to provide
energy transfer from the donor to the acceptor through the
well-known Foerster mechanism. Thus, the distance between the two
fluorophores as determined by the number of atoms in the chain
separating the two fluorophores can be varied in accordance with
the nature of the chain. Various chains or backbones may be
employed, such as nucleic acids, both DNA and RNA, modified nucleic
acids, e.g. where oxygens may be substituted by sulfur, carbon, or
nitrogen, phosphates substituted by sulfate or carboxylate, etc.,
polypeptides, polysaccharides, various groups which may be added
stepwise, such as di-functional groups, e.g. haloamines, or the
like. The fluorophores may be substituted as appropriate by
appropriate functionalization of the various building blocks, where
the fluorophore may be present on the building block during the
formation of the label, or may be added subsequently, as
appropriate. Various conventional chemistries may be employed to
ensure that the appropriate spacing between the two fluorophores is
obtained.
The molecular weights of the labels (fluorophores plus the backbone
to which they are linked) will generally be at least about 250 Dal
and not more than about 10,000 Dal, usually not more than about
8,000 Dal. The molecular weight of the fluorophore will generally
be in the range of about 250 to 2,000 Dal, where the molecular
weights of the acceptor-donor pairs on different labels to be used
together will usually not differ by more than about 20%. The
fluorophores may be bound internal to the chain, at the termini, or
one at one terminus and another at an internal site. The
fluorophores may be selected so as to be from a similar chemical
family, such as cyanine dyes, xanthenes or the like. Thus, one
could have the donors from the same chemical family, each
donor-acceptor pair from the same chemical family or each acceptor
from the same family or the combination from a different
family.
The subject labels find particular application in various
separation techniques, such as electrophoresis, chromatography, or
the like, where one wishes to have optimized spectroscopic
properties, high sensitivity and comparable influence of the labels
on the migratory aptitude of the components being analyzed. Of
particular interest is electrophoresis, such as gel, capillary,
etc. Among chromatographic techniques are HPLC, affinity
chromatography, thin layer chromatography, paper chromatography,
and the like.
It is found that the spacing between the two fluorophores will
affect the mobility of the label. Therefore, one can use different
dye pairs and by varying the distance between the different dye
pairs, within a range which still permits good energy transfer,
provide for substantially constant mobility for the labels. The
mobility is generally not systematically related to the specific
spacing, so that one will empirically determine the effect of the
spacing on the mobility of a particular label. However, because of
the flexibility in the spacing of the fluorophores in the labels,
by synthesizing a few different labels with different spacings and
different dye pairs, one can now provide for a family of
fluorescent labels, which share a common excitation, that have
strong and distinctive emission and a substantially common
mobility. Usually, the mobility will differ by not more than about
20% of each other, preferably not more than about 10% of each
other, and more preferably within about 5% of each other, when used
in a particular separation. Generally, this will translate to less
than about 1 base difference, preferably not more than about 0.5
base difference. The mobility may usually be determined by carrying
out the separation of the labels by themselves or the labels bound
to a common molecule which is relevant to the particular
separation, e.g. a nucleic acid molecule of the appropriate size,
where one is interested in sequencing.
A wide variety of fluorescent dyes may find application. These dyes
will fall into various classes, where combinations of dyes may be
used within the same class or between different classes. Included
among the classes are dyes, such as the xanthene dyes, e.g.
fluoresceins and rhodamines, coumarins, e.g. umbelliferone,
benzimide dyes, e.g. Hoechst 33258, phenanthridine dyes, e.g. Texas
Red, and ethidium dyes, acridine dyes, cyanine dyes, such as
thiazole orange, thiazole blue, Cy 5, and Cyfr, carbazole dyes,
phenoxazine dyes, porphyrin dyes, quinoline dyes, or the like.
Thus, the dyes may absorb in the ultraviolet, visible or infra-red
ranges. For the most part, the fluorescent molecules will have a
molecular weight of less than about 2 kDal, generally less than
about 1.5 kDal.
The energy donor should have strong molar absorbance coefficient at
the desired excitation wavelength, desirably greater than about
10.sup.4, preferably greater than about 10.sup.5 cm.sup.-1
M.sup.-1. The excitation maximum of the donor and the emission
maximum of the acceptor (fluorescer) will be separated by at least
15 nm or greater. The spectral overlap integral between the
emission spectrum of the donor chromophore and the absorption
spectrum of the acceptor chromophore and the distance between the
chromophores will be such that the efficiency of energy transfer
from donor to acceptor will range from 20% to 100%.
Separation of the donor and acceptor based on number of atoms in
the chain will vary depending on the nature of the backbone,
whether rigid or flexible, involving ring structures or non-cyclic
structures or the like. Generally the number of atoms in the chain
(the atoms in the ring structures will be counted as the lowest
number of atoms around one side of the ring for inclusion in the
chain) will be below about 300, usually below about 200 atoms,
preferably below about 150, where the nature of the backbone will
influence the efficiency of energy transfer between donor and
acceptor. Conveniently, one of the dyes may be on the 5' or 3'
terminal nucleoside.
While for the most part, pairs of fluorophores will be used, there
can be situations where up to four different, usually not more than
three different, fluorophores bound to the same backbone may find
use. By using more fluorophores, one may greatly extend the Stokes
shift, so that one may excite in the visible wavelength range and
have emission in the infra-red wavelength range, usually below
about 1000 nm, more usually below about 900 nm. Detecting light in
the infra-red wavelength range has many advantages, since it will
not be subject to interference from Raman and Rayleigh light
resulting from the excitation light. In order to maintain the
mobility constant, one may use the same number of fluorophores on
the labels, having a multiplicity of the same fluorophore to match
the number of fluorophores on labels having different fluorophores
for the large Stokes shift.
The subject invention finds particular application with nucleic
acid chains, where the nucleic acid chains find use as primers in
sequencing, the polymerase chain reaction, particularly for sizing,
or other system where primers are employed for nucleic acid
extension and one wishes to distinguish between various components
of the mixture as related to the particular labels. For example, in
sequencing, universal primers may be employed, where a different
pair of fluorophores are used for each of the different
dideoxynucleosides used for the extension during sequencing
reactions.
A large number of nucleosides are available, which are
functionalized, and may be used in the synthesis of a
polynucleotide. By synthesizing the subject nucleic acid labels,
one can define the specific sites at which the fluorophores are
present. Commercially available synthesizers may be employed in
accordance with conventional ways, so that any sequence can be
achieved, with the pair of fluorophores having the appropriate
spacing.
As already indicated, the subject labels find particular use in
sequencing. For example, universal primers may be prepared, where
the primer may be any one of the universal primers, having been
modified by bonding of the two fluorophores to the primer. Thus,
various commercial primers are available, such as primers from
pUC/M13, .lambda.gt10, .lambda.gt11, and the like. See, Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd ed., CSHL, 1989,
Section 13. DNA sequences are cloned in an appropriate vector
having a primer sequence joined to the sequence to be sequenced.
Different 2', 3' ddNTPs are employed, so that termination occurs at
different sites, depending upon the particular ddNTP which is
present in the chain extension. By employing the subject primers,
each ddNTP will be associated with a particular label. After
extension with the Klenow fragment, the resulting fragments may
then be separated in a single lane by electrophoresis or in a
single capillary by electrophoresis, where one can detect the
terminating nucleotide by virtue of the fluorescence of the
label.
Kits are provided having combinations of labels, usually at least
2. Each of the labels will have the acceptor-donor pair, usually
with comparable backbones, where the labels will be separated along
the backbone to give comparable mobility in the separation method
to be used. Each of the labels in a group to be used together will
absorb at about the same wavelength and emit at different
wavelengths. Each of the labels in the group will have about the
same effect on mobility in the separation method, as a result of
the variation in placement of the different fluorophores along the
backbone.
The kits will generally have up to about 6, usually about up to
about 4 different labels which are matching, but may have 2 or more
sets of matching labels, having 2-6 different labels.
Of particular interest are labels comprising a nucleic acid
backbone, where the labels will generally have at least about 10
nucleotides and not more than about 50 nucleotides, usually not
more than about 30 nucleotides. The labels may be present on the
nucleotides which hybridize to the complementary sequence or may be
separated from those nucleotides. The fluorophores will usually be
joined to the nucleotide by a convenient linking arm of from about
2 to 20, usually 4 to 16 atoms in the chain. The chain may have a
plurality of functionalities, particularly non-oxo-carbonyl, more
particularly ester and amide, amino, oxy, and the like. The chain
may be aliphatic, alicyclic, aromatic, heterocyclic, or
combinations thereof, usually comprising carbon, nitrogen, oxygen,
sulfur, or the like in the chain.
The entire nucleic acid sequence may be complementary to the 5'
primer sequence or may be complementary only to the 3' portion of
the sequence. Usually, there will be at least about 4 nucleotides,
more usually at least about 5 nucleotides which are complementary
to the sequence to be recognized. The primers are combined with the
sequence to be recognized in the appropriate plasmid having the
primer sequence at the 3' end of the strand to be copied and dNTPs
added with a small amount of the appropriate ddNTP. After
extension, the DNA may be isolated and transferred to a gel or
capillary for separation.
The kits which are employed will have at least two of the subject
labels, which will be matched by having substantially the same
absorption for the donor molecule, distinct emission spectra and
substantially the same mobility. Generally for single stranded
nucleic acids, the separation will be from about 3-20, more usually
3-15, preferably about 3-10 nucleosides between fluorophores.
The following examples are offered by way of illustration and not
by way of limitation.
EXPERIMENTAL
Design and synthesis of ET primers. The structures of the ET
primers and a representative synthetic reaction are presented in
FIG. 1. Oligodeoxynucleotides (18-bases long) with the sequence
5'-GTTTTCCCAGTCACGACG-3' (SEQ ID NO:01) (the M13-40 universal
primer) were synthesized with donor-acceptor fluorophore pairs
separated by different distances. The 18-mer contains a modified
base (T*) introduced by the use of
5'-dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2'-deoxy
uridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
(Amino-Modifier C6 dT, Glen Research, Sterling, Va.) (Structure 1),
which has a protected primary ##STR1##
Structure 1. Amino Modifier C6 dT
amine linker arm. The donor dye was attached to the 5' end of the
oligomer, and the acceptor dye was attached to the primary amine
group on the modified base (T*). The ET primers are named using the
abbreviation D-N-A, where D is the donor, A is the acceptor, and N
is the number of bases between D and A. In all the primers
prepared, 5-carboxyfluorescein (FAM, F) is selected as a common
donor, 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE,
J), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA, T) and
6-carboxy-X-rhodamine (ROX, R) are selected respectively as
acceptors. As a representative example, the structure of F3T is
shown below (Structure 2). ##STR2##
Structure 2. F3T
To prepare the ET primers, the donor FAM was introduced by using
6-FAM amidite in the last step of the oligonucleotide synthesis on
a DNA synthesizer. After cleavage from the solid support and
removal of the base protecting groups, the primers were evaporated
to dryness under vacuum (0.5 mm Hg). To incorporate the acceptor
dyes, 15-20 nmol of FAM-labeled T*-containing oligonucleotides in
40 .mu.l 0.5M Na.sub.2 CO.sub.3 /NaHCO (pH 9.0) buffer were
incubated overnight at room temperature with an approximately
150-fold excess of corresponding FAM, JOE, TAMRA and ROX
N-hydroxysuccinimidyl esters in 12 .mu.l DMSO. Unreacted dye was
removed by size exclusion chromatography on a Sephadex G-25 column
(Pharmacia, Piscataway, N.J.). The ET primers were then purified by
electrophoresis in a 20% polyacrylamide gel containing 6M urea (40
cm.times.0.8 cm). The purified primers were recovered from the gel
slices and desalted with Oligonucleotide Purification Cartridge
(Applied Biosystems, Foster city, Calif.). The single dye-labeled
primers with the same sequence as that of the ET primers were
prepared by the standard protocol using Aminolink 2 (Applied
Biosystems, Foster city, Calif.). The purity of the primers was
shown to be >99% by polyacrylamide capillary gel
electrophoresis. Primers were quantified by their 260 nm
absorbances and then stored in 10 mM Tris-Cl, 1 mM EDTA (pH 8.0) at
a final concentration of 0.4 pmol/.mu.l for DNA sequencing
reactions.
Twenty ET primers were synthesized with the same donor at 5' end
and different acceptors at different positions on the primer
sequence. The spacing between the two chromophores is altered by
varying the position of T* in the synthesis of each primer. We
found that the electrophoretic mobility of the ET primers depends
on the spacing between the donor and acceptor. Within a range of
distances determined by the number of intervening bases that allow
good energy transfer, it is possible to adjust the electrophoretic
mobility of the primers. The advantages of the energy transfer
approach described here are (1) that a large Stokes shift and much
stronger fluorescence signals can be generated when exciting at 488
nm and (2) that the mobility of the primers can be tuned by varying
the distances between the donor and acceptor to achieve the same
mobility. As a representative example, FIG. 2A, 2B, 2C, and 2D
present the absorption and emission spectra of the ET primer F10F,
F10J, F3T and F3R respectively. Each ET primer exhibits the
characteristic absorption of FAM at 496 nm as well as strong
absorption at 525 nm due to JOE in F10J, at 555 nm due to TAMRA in
F3T and at 585 nm due to ROX in F3R. The fluorescence spectra of
the ET primers are dominated by the acceptor emissions. While the
emission maximum of F10F is at 525 nm, the emission of F10J with
488-nm excitation is Stokes-shifted to 555 nm, that of F3T is
shifted to 580 nm, and that of F3R is shifted to 605 nm. In the
case of F3R, the Stokes shift is over 100 nm. FIGS. 2A, 2B, 2C, and
2D also presents emission spectra of the single dye-labeled primers
measured at the same molar concentration as that of the
corresponding ET primers. Substantial enhancement of the ET primer
emission intensity is observed compared to the corresponding single
dye-labeled primers, indicating that efficient energy transfer is
occurring. The fluorescence intensity improvements derived from
FIGS. 2A, 2B, 2C, and 2D are: F10F=1.8.times.FAM;
F10J=2.5.times.JOE or 1.4.times.JOE when JOE is excited at 514 nm;
F3T=5.3.times.TAMRA or 1.7.times.TAMRA when TAMRA is excited at 514
nm; F3R=6.2.times.ROX or 2.3.times.ROX when ROX is excited at 514
nm. Thus, the fluorescence intensity of single JOE, TAMRA and ROX
labeled primer with 514 nm excitation is still less than that of
the corresponding ET primer with 488 nm excitation. To evaluate the
emission spectral purity of the four ET primers, their normalized
emission spectra are presented in FIG. 3. It can be seen that the
residual emission of FAM in F10J, F3T and F3R is very small. Based
on a comparison of the residual FAM emission in the ET primers with
that of a FAM-labeled primer with same sequence and length, the
energy transfer efficiency was calculated to be 65% for F10J, 96%
for F3R and 97% for F3T. FIGS. 4A, 4B, 4C, and 4D presents the
fluorescence intensity comparison of the ET primer series as well
as the corresponding single dye-labeled primers measured at the
same molar concentration. The results indicate that when the two
fluorophores are too close to each other, fluorescence quenching
occurs. The fluorescence intensity increases with the increase of
the separation distances between the donor and acceptor. Strong
fluorescence signals were obtained when the separation distance is
10-bases. The fluorescence intensity of F10T and F10R measured at
the acceptor emission region is 10 and 14 times that of TAMRA and
ROX primer respectively. Thus, the maximum fluorescence signals can
be increased as much as 14-fold using the ET principle. The results
also indicate that the donor FAM emission intensity in F10T and
F10R is higher than the other ET primers. However, for a particular
primer, as long as the acceptor emission is higher than or equal to
that of the donor and the net fluorescence signal is intense, it is
valuable for DNA analysis. The mobility comparison of ET primers on
polyacrylamide capillary electrophoresis are shown in FIG. 5 which
indicates that F10F, F10J, F10R, F3T and F3R have very similar
mobility shifts. Although F10T has large mobility difference
compared to F10F, F10J and F10R, FIG. 6 shows that the extended
ddAPT/dNTPs DNA fragments generated with F10T have similar
mobilities as those generated with F10F, F10J and F10R. This
indicates that as the DNA fragments grow longer than 18 bases, DNA
fragments generated with F10T have essentially the same
conformation as fragments generated with F10F, F10J and F10R. For
the successful application of donor-acceptor fluorophore labeled
primers to DNA sequencing, it is useful that the primers produce
same mobility shifts of the DNA fragments and display distinct
fluorescence signals. Six primers (F10F, F10J, F10T, F10R, F3T and
F3R) were therefore selected for evaluation in DNA sequencing.
DNA Sequencing procedure. Sequencing was performed using M13mp18
template DNA and modified T7 DNA polymerase on a 4-color capillary
electrophoresis (CE) DNA sequencer designed in our laboratory and
on an ABI 373A sequencer. Four reactions were run, one for each
dye/ddNTP combination. The reactions containing ddCTP were run with
the F10F primer, ddATP with the F10J primer, ddGTP with the F3T or
F10T primer, and ddTTP with the F3R or F10R primer. The working
buffer was prepared by freshly mixing equal volumes of 400 mM MOPS
(pH 7.5), 500 mM NaCl, 100 mM MgCl.sub.2 (10.times.MOPS buffer) and
50 mM MnCl.sub.2, 150 mM sodium isocitrate (10.times.Mn buffer).
One .mu.l of this buffer was then combined with 1.mu.l of primer
(0.4 pmol), the indicated amount of template DNA, and water to a
total volume of 5 .mu.l. The mixtures were annealed by heating at
65.degree. C. for 2 min and slowly (.about.35 min) cooling to
<30.degree. C. Three .mu.l of dNTP/ddNTP mix (2.4 mM each of
dGTP, dATP, dTTP and dCTP with 8 .mu.M of the specific ddNTP) were
then added and the reaction mixture warmed to 37.degree. C. for 2
min. Then 2 .mu.l of a freshly diluted mixture of T7 DNA polymerase
(2 units/.mu.l) and yeast pyrophosphatase (1.5 units/ml) were added
and incubation continued at 37.degree. C. for 30 min. The four
reaction mixtures for each sequence were then stopped and combined
into one vial and 4 .mu.l of 3M sodium acetate and 180 .mu.l of 95%
ethanol were added to precipitate the DNA fragments. After 15 min
at -20.degree. C., the precipitated DNA was collected by
centrifugation (12,000 .times.g) for 15 min, and washed twice with
70% ethanol. The precipitated DNA was vacuum dried, and resuspended
in 2 .mu.l 98% formamide containing 1 mM EDTA (for CE sequencer) or
5 .mu.l of deionized formamide containing 8.3 mM EDTA (for ABI
sequencer) and heated at 95.degree. C. for 2 min. For CE sequencer,
samples were introduced electrophoretically into a 65 cm long (45
cm effective length) 3%T and 3%C polyacrylamide gel filled
capillary. Electrophoresis was run at 150 V/cm. One argon laser at
488 nm is used for excitation and the fluorescence signals were
collected in four channels centered at 525, 555, 580 and >610
nm. For ABI sequencer, the denatured DNA was loaded on a 6%
polyacrylamide 8.3M urea denaturing gel mounted in the instrument.
Electrophoresis was conducted at a constant power of 35 W for 12-14
hours using Tris-taurine-EDTA buffer. The data were analyzed using
the ABI 373A software (version 1.2.0).
DNA sequencing results with ET primers. DNA sequencing using primer
F10F, F10J, F10T and F10R on CE sequencer was performed using 0.4
pmol of primer and 0.2 .mu.g of template DNA for each base
extension. The sequences extended to more than 600 bases, a portion
of which (raw data) is shown in FIG. 7. From this raw data,
sequences can be determined by the color on the top peak of the
electropherograms. This is the first 4-color sequencing plot
without any mobility shift adjustment.
ET primers described here also provide better results and higher
sensitivity on the commercial 4-color DNA sequencer. To demonstrate
the advantage of ET primers versus conventional single-dye labeled
printers. DNA sequencing samples generated with primer F10F, F3T
and F3R were analyzed on an Applied Biosystems 373A sequencer.
Single base extension (ddTTP/dNTPs) experiments were performed to
examine the relative mobility shift and sensitivity of DNA
fragments generated with the ET primers. FIGS. 8A, 8B, 8C, 8D, and
8E present raw fluorescence intensity traces from electrophoresis
run on an ABI 373A sequencer. The graphs in FIG. 8A were obtained
using M13 (-40) primers labeled with single dye molecules. The
differences in electrophoretic mobility of the DNA fragments can be
clearly seen. The TAMRA- and ROX-labeled fragments migrate about
one base slower than the FAM- and JOE-tagged DNA fragments and have
dramatically weaker fluorescence intensities. The corresponding
runs with the ET primers are presented in FIG. 8B. The mobilities
of the DNA fragments are more closely matched (less than a quarter
of a base difference).
To further quantify the instrument sensitivity with the ET primers
under slab gel conditions, reactions were run using a constant
amount of primer (0.4 pmol) and varying the amount of M13mp18
template DNA (0.05-1 pmol). Graphs of several band intensities
against quantity of template were made. This method indicates that
the sensitivity for the F10F primer is 160% that of the FAM primer.
Similarly, the sensitivity for the F10J, F3T and F3R primers is
360%, 400% and 470% that of JOE, TAMRA and ROX primers,
respectively. In experiments which included an excess of template
DNA over primer, only a small fraction of either ET or single-dye
labeled primer remained unextended. Thus, no significant difference
was seen in the efficiency with which the ET primers were extended
by polymerase compared with single-dye labeled primers.
Typical raw fluorescence intensity traces for 4-dye, single lane
sequences are presented in FIGS. 8C and 8D. Shown here is a portion
from the middle of the run spanning about 45 bases. On this
intensity scale, the peaks from the red filter are barely
discernible when single ROX-labeled primer is used (c). In
contrast, all of the sequence-dependent intensity fluctuations are
readily seen with the ET primers in the raw data (D). While
four-color sequences run with this instrument typically require
3-fold more template and 2-fold more primer in the reactions
containing TAMRA- and ROX-labeled primers, the four reactions used
for FIG. 8D contained equal amounts of ET primer and template. This
change in reaction balance was made possible by the increased
relative intensities of the F3T and F3R primers. With these four
primers, DNA sequencing on M13Mp18 template produces 510 bases with
accuracy of over 99.8%. This sequence can be obtained using a total
of 0.6 .mu.g (0.24 pmol) of M13 template DNA which is approximately
one-fourth the amount of template DNA required to give similar
sequence accuracy with single dye-labeled primers.
It is evident from the above results, that one can tune related
compositions, e.g. polynucleotides functionalized with 2
fluorophores to provide for different emission wavelengths and high
emission quantum yields, while having substantially the same
excitation-light absorbance and mobility. In this way, mixtures of
compositions may be independently analyzed, where the different
components may be differentially labeled with labels having
differing fluorescence emission bands. Furthermore, the
compositions can be readily prepared, can be used in a wide variety
of contexts, and have good stability and enhanced fluorescent
properties.
All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 1 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
other nucleic acid (A) DESCRIPTION: /desc ="Synthesized Primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GTTTTCCCAGTCACGACG18
__________________________________________________________________________
* * * * *