U.S. patent number 5,952,180 [Application Number 09/080,940] was granted by the patent office on 1999-09-14 for sets of labeled energy transfer fluorescent primers and their use in multi component analysis.
This patent grant is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Jingyue Ju.
United States Patent |
5,952,180 |
Ju |
September 14, 1999 |
Sets of labeled energy transfer fluorescent primers and their use
in multi component analysis
Abstract
Sets of fluorescent energy transfer labels and methods for their
use in multi component analysis, particularly nucleic acid
enzymatic sequencing, are provided. In the subject sets, at least
two of the labels are energy transfer labels comprising a common
donor and acceptor fluorophore in energy transfer relationship
separated by different distances and capable of providing
distinguishable fluorescence emission patterns upon excitation at a
common wavelength. The subject labels find particular use in a
variety of multi-component analysis applications, such as probes in
FISH and multi array analyses, as well as primers in nucleic acid
enzymatic sequencing applications.
Inventors: |
Ju; Jingyue (Redwood City,
CA) |
Assignee: |
Incyte Pharmaceuticals, Inc.
(Palo Alto, CA)
|
Family
ID: |
25131547 |
Appl.
No.: |
09/080,940 |
Filed: |
May 19, 1998 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
968327 |
Nov 12, 1997 |
5814454 |
|
|
|
784162 |
Jan 15, 1997 |
5804386 |
|
|
|
Current U.S.
Class: |
435/6.12;
435/91.2 |
Current CPC
Class: |
C12Q
1/6869 (20130101); C12Q 1/6818 (20130101); C12Q
1/6818 (20130101); C12Q 2565/1015 (20130101); C12Q
2537/143 (20130101); C12Q 2525/207 (20130101); C12Q
1/6869 (20130101); C12Q 2565/1015 (20130101); C12Q
2525/207 (20130101); C12Q 2525/185 (20130101); C12Q
1/6869 (20130101); C12Q 2565/1015 (20130101); C12Q
1/6869 (20130101); C12Q 2563/113 (20130101) |
Current International
Class: |
C12P
19/00 (20060101); C12P 19/34 (20060101); C12Q
1/68 (20060101); C12Q 001/68 (); C12P 019/34 () |
Field of
Search: |
;435/6,91.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO91/19735 |
|
Dec 1991 |
|
WO |
|
95/21266 |
|
Aug 1995 |
|
WO |
|
Other References
Ansorge, W., (1987) Automated DNA sequencing: ultrasensitive
detection of fluorescent bands during electrophoresis, Nucl. Acids
Res. 15 (11): 4593-4602. .
Glazer, A., et al., (1983) "Fluorescent Tandem Phycobiliprotein
Conjugates," Biophys. S. 43:383-386. .
Griflin, H., et al., (1993) "DNA Sequencing--Recent Innovations and
Future Trends," Appl. Biochem. and Biotech. 38:147-159. .
Huang, X, et al., 1992 "DNA Sequencing Using Capillary Array
Electrophoresis," Anal. Chem. 64: 2149-2154. .
Ju, J., et al., (1995) "Design and Synthesis of Fluorescence Energy
Transfer Dye-Labeled Primers and Their Application for DNA
Sequencing and Analysis," Anal. Bio. 231:131-140. .
Ju, J., et al., (1995) "Fluorescence energy transfer dye-labeled
primers for DNA sequencing and analysis," Proc. Natl. Acad. Sci.
USA 92: 4347-4351. .
Ju, J., et al., (1996) "Energy transfer primers: A new fluorescence
labeling paradigm for DNA sequencing and analysis," Nature Medicine
2(2): 246-249. .
Lu, H., et al., (1994) "High-speed and high-accuracy DNA sequencing
by capillary gel electrophoresis in a simple, Low cost instrument
Two-color peak-height encoded sequencing at 40.degree.C" J.
Chromatography A 680:497-501. .
Matteucci, M., et al., (1996) "In pursuit of antisense," Nature
384: 20-22. .
Prober, J., et al., (1987) "A System for Rapid DNA Sequencing with
Fluorescent Chain-Terminating Didcoxynucleotides," Science
238:336-341. .
Repp, R., et al., (1995) "Detection of Four Different 11q23
Chromosomal Abnormalities by Multiplex-PCR and Fluorescence-Based
Automatic DNA-Fragment Analysis," Leukemia 9: 210-215. .
Schena, M., et al., (1995) "Quantitaitive Monitoring of Gene
Expression Patterns with a Complementary DNA Microarray," Science
270: 467-469. .
Smith, L., et al., (1986) "Fluorescence detection in automated DNA
sequence analysis," Nature 321:674-679. .
Wang, Y., et al., (1995) "Rapid Sizing of Short Tandem Repeat
Alleles Using Capillary Array Electrophoresis and Energy-Transfer
Fluorescent Primers," Anal. Chem. 67(7): 1197-1203. .
Ziegle, J., et al., (1992) "Application of Automated DNA Sizing
Technology for Genotyping Microsatellite Loci," Genomics, 14:
1026-1031. .
Ju, Jingyue, et al., "Cassette labeling for facile construction of
energy transfer fluorescent primers," Nucleic Acids Research (1996)
vol. 24. No.(6):1144-1148..
|
Primary Examiner: Campbell; Eggerton A.
Attorney, Agent or Firm: Bozicevic, Field& Francis
Field; Bret
Parent Case Text
This application is a continuation of Ser. No. 08/968,327, filed
Nov. 12, 1997, now U.S. Pat. No. 5,814,454 which is a divisional of
Ser. No. 08/784,162, filed Jan. 15, 1997, now U.S. Pat. No.
5,804,386.
Claims
What is claimed is:
1. A method of sequencing a DNA molecule, said method
comprising:
enzymatically producing four sets of primer extension products
using said DNA molecule as a template, wherein each of said sets
comprises a family of different sized primer extension products
terminating at the same base, and further wherein each of said sets
comprises the same fluorescent label, wherein the fluorescent
labels from at least two of said sets comprise the same donor and
acceptor fluorophores in energy transfer relationship separated by
different distances and capable of providing distinguishable
fluorescence emission patterns,
size separating said primer extension products;
detecting said size separated primer extension products; and
determining said sequence from said detected size separated primer
extension products.
2. The method according to claim 1, wherein said detecting
comprises irradiating said primer extension products at a common
wavelength and distinguishing said distinguishable fluorescence
emission patterns.
3. The method according to claim 2, wherein said method further
comprises producing an electropherogram plotting the emission
intensity at two wavelengths of each of said primer extension
products as a function of time and said sequence is derived from
said electropherogram.
4. A method of sequencing a DNA molecule, said method
comprising:
enzymatically producing a first set of primer extension products
terminating in A using a first energy transfer fluorescently
labeled oligonucleotide primer comprising a donor fluorophore and
an acceptor fluorophore in energy transfer relationship separated
by a distance x;
enzymatically producing a second set of primer extension products
terminating in G using a second energy transfer fluorescently
labeled oligonucleotide primer comprising said donor fluorophore
and said acceptor fluorophore in energy transfer relationship
separated by a distance y;
enzymatically producing a third set of primer extension products
terminating in T using a third energy transfer fluorescently
labeled oligonucleotide primer comprising said donor fluorophore
and said acceptor fluorophore in energy transfer relationship
separated by a distance z; and
enzymatically producing a fourth set of primer extension products
terminating in C using a fourth energy transfer fluorescently
labeled oligonucleotide primer wherein said donor and acceptor
fluorophore are the same;
wherein said first through fourth fluorescent labels are capable of
providing distinguishable fluorescence emission patterns;
combining said four sets of primer extension products;
size separating said primer extension products;
irradiating said size separated primer extension products whereby
each of said side separated product emits a distinguishable
fluorescence emission pattern;
producing an electropherogram by plotting the intensity of emitted
light at two wavelengths as a function of time as each primer
extension product passes relative to a detector; and
determining said sequence of said DNA molecule from said
electropherogram.
5. A method of determining the presence of at least two different
components in a sample, said method comprising:
labeling a first component of said sample with a first fluorescent
label comprising at least one donor and at least one acceptor
fluorescer component separated by a distance X;
labeling a second component of said sample with a second
fluorescent label comprising said at least one donor and said at
least one acceptor fluorescer components separated by a distance Y,
wherein Y is different from X;
irradiating said sample with light at a wavelength absorbed by said
acceptor fluorescer component, whereby said first and second labels
emit distinguishable fluorescence emission patterns;
detecting said distinguishable fluorescence emission patterns;
and
determining the presence of said two different components from said
detection.
6. The method according to claim 5, wherein said first and second
fluorescent labels are energy transfer labeled oligonucleotide
primers and said first and second components are primer extension
products comprising said primers.
7. The method according to claim 5, wherein said first and second
fluorescent labels are energy transfer dye labeled probes and said
first and second components are targets to which specifically bind
to said probes.
8. A kit for use in DNA sequencing, said kit comprising:
a set of four fluorescently labeled oligonucleotide primers,
wherein at least two of said oligonucleotide primers have a common
donor and acceptor fluorophore in energy transfer relationship and
are separated by different distances.
9. The kit according to claim 8, wherein said kit further comprises
polymerase.
10. The kit according to claim 8, wherein said kit further
comprises deoxynucleotides and dideoxynucleotides.
Description
TECHNICAL FIELD
The field of this invention is fluorescent labels, particularly
fluorescently labeled primers for use in DNA sequencing
applications.
BACKGROUND OF THE INVENTION
Fluorescent labels find use in variety of different biological,
chemical, medical and biotechnological applications. One example of
where such labels find use is in polynucleotide sequencing,
particularly in automated DNA sequencing, which is becoming of
critical importance to large scale DNA sequencing projects, such as
the Human Genome Project.
In methods of automated DNA sequencing, differently sized
fluorescently labeled DNA fragments which terminate at each base in
the sequence are enzymatically produced using the DNA to be
sequenced as a template. Each group of fragments corresponding to
termination at one of the four labeled bases are labeled with the
same label. Thus, those fragments terminating in A are labeled with
a first label, while those terminating in G, C and T are labeled
with second, third and fourth labels respectively. The labeled
fragments are then separated by size in an electrophoretic medium
and an electropherogram is generated, from which the DNA sequence
is determined.
As method of automated DNA sequencing have become more advanced, of
increasing interest is the use of sets of fluorescent labels in
which all of the labels are excited at a common wavelength and yet
emit one of four different detectable signals, one for each of the
four different bases. Such labels provide for a number of
advantages, including high fluorescence signals and the ability to
electrophoretically separate all of the labeled fragments in a
single lane of an electrophoretic medium which avoids problems
associated with lane to lane mobility variation.
Although such sets of labels have been developed for use in
automated DNA sequencing applications, heretofore the differently
labeled members of such sets have each emitted at a different
wavelength. Thus, conventional automated detection devices
currently employed in methods in which all of the enzymatically
produced fragments or primer extension products are separated in
the same lane must be able to detect emitted fluorescent light at
four different wavelengths. This requirement can prove to be an
undesirable limitation. More specifically, carrying out sequencing
on vast numbers of different DNA templates simultaneously increases
the number of different fragments and corresponding labels
required. At the same time, there is a need for a reduction in the
complexity of the detection device, e.g. a device which can operate
with light detection at only two wavelengths is preferable.
It would therefore be desirable to develop sets of fluorescent
labels capable of providing four distinguishable signals, where the
number of wavelengths associated with the four different signals is
less than the number of different labels, e.g. where four different
labels provide signals comprising emitted light at from one to two
wavelengths. With such sets one could either: (1) reduce the
complexity of automated detector devices or (2) increase the
throughput of detectors capable of detecting at four different
wavelengths, thereby achieving sequencing two DNA templates, or the
same double stranded templates from both the 5' and 3' end,
simultaneously.
Relevant Literature
DNA sequencing is reviewed in Griffin & Griffin, Appl. Biochem.
Biotechnol. (1993) 38: 147-159. Fluorescence energy transfer labels
and their use in DNA sequencing applications are described in Ju et
al., Nucleic Acids Res. (1996) 24: 1144-1148, Ju et al., Nat. Med.
(1996) 2: 246-249, Ju et al., Anal. Biochem. (1995) 231: 131-140.
Ju et al., Proc. Natl. Acad. Sci. USA (1995) 92: 4347-4351. Use of
fluorescent energy transfer labels for non-DNA sequencing multi
component analysis application is described in Wang et al., Anal.
Chem. (1995) 67: 1197-1203; Ziegle et al., Genomics (1992) 14:
1026-1031; and Repp et al., Leukemia (1995) 9: 210-215. Other
references describing multi-component analysis applications include
Schena et al., Science (1995) 270: 467-469.
Other references of interest include U.S. Pat. Nos. 4,996.143 and
5,326,692, as well as Glazer and Streyer, Biophys. J. (1983) 43:
383-386, Huang et al., Anal. Chem. (1992) 64: 2149-2154; Prober et
al., Science ( 1987) 336-341; Smith et al., Nature (1986) 321:
674-679, Lu et al, J. Chromat. A (1994) 680: 497-501 and Ansorge et
al., Nucleic Acids Res. (1987) 15: 4593-4603.
SUMMARY OF THE INVENTION
Sets of fluorescent labels, particularly labeled primers, as well
as methods for their use in multi component analysis, are provided.
At least two of the labels of the subject sets comprise a common
donor and acceptor fluorescer component in energy transfer
relationship separated by different distances, such that the labels
provide distinguishable fluorescent signals upon excitation at a
common light wavelength. The subject sets of labels find use in a
variety of applications requiring a plurality of distinguishable
fluorescent labels, and find particular use as primers in nucleic
acid enzymatic sequencing applications. Primers with the same
labels which produce distinguishable emission patterns can be
produced because energy transfer between the acceptor and donor
fluorphores is a function of the separation distance between the
acceptor and donor in the label. By changing the distance,
different fluorescence emission patterns are obtained.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the general labeling concept using four fluorescent
molecules to generate at least eight fluorescent dye-labeled
primers with distinguishable fluorescence emission patterns.
FIG. 2 shows the structure of the four primers labeled with two
different fluorescent dyes, 6-carboxyfluoroscein (FAM, F) as a
donor and 6-carboxyrhodamine (ROX, R) as an acceptor. The numbers
in the primer name indicate the intervening nucleotides between the
donor and acceptor.
FIG. 3 shows that the fluorescence signal of the four fluorescent
primers is sufficiently different to code for four nucleotides T
(F6R), G (F13R), A (F16R), C (F16F). It also shows that the primer
F16T using 6-carboxy tetramethyl rhodamine (TAMRA, T) to replace
ROX (R) as an acceptor displays almost equal fluorescence signal
intensity of F (blue) and T (black). The fluorescence signals shown
are the electropherograms of the single base extension fragments
from each primer obtained in the ABI four color fluorescent 377 DNA
sequencer which has the appropriate filters to detect fluorescence
signals from FAM (F .lambda..sub.em(max) =525 nm), ROX (R
.lambda..sub.em(max) =605 nm) and TAMRA (T .lambda..sub.em(max)
=580 nm).
FIG. 4 shows that the fluorescence intensity of the single base
extension fragments from primer F6R (T fragments) and F13R (G
fragments) due to energy transfer from F to R is much higher than
that of the single base fragments generated with primer R15R (T
fragments) which carries two ROX dyes but with same sequence as F6R
and F13R. Same concentration of the primer and other sequencing
reagents were used in the comparison.
FIG. 5 shows a small portion of the raw sequencing data in 2-color
mode (FAM, F .lambda..sub.em(max) =525 nm, blue; ROX, R
.lambda..sub.em(max) =605 nm, red) generated by primer F6R (T),
F13R (G), F16R (A), F16F (C) and a cDNA clone which has a polyA
tail at the 3' end. Sequences can be called by the color patterns
of each peak.
FIGS. 6A and 6B shows large portion of the raw sequencing data
(from nucleotide 30 to 130) in 2-color mode (FAM,F
.lambda..sub.em(max) =525 nm, blue; ROX, R .lambda..sub.em(max)
=605 nm. red) generated by primer F6R (T). F13R (G). F16R (A), F16F
(C) and a cDNA clone which has a polyA tail at the 3' end.
Sequences can be called by the color patterns of each peak. Samples
were prepared using Thermo Sequenase Kit (Amersharn LIFE SCIENCE)
and run on a ABI 377 DNA sequencer with virtual filter A that
detects the fluorescence signal from FAM and ROX.
FIG. 7 is a shematic of the Sanger enzymatic DNA sequencing
method.
DEFINITIONS
The term "fluorescent label" refers to a compound comprising at
least one fluorophore bonded to a polymer.
The term "energy transfer fluorescent label" refers to a compound
comprising at least two fluorophores in energy transfer
relationship, where the fluorophores are bonded to a spacer
component, e.g. a polymeric moiety, which separates the two
fluorphores by a certain distance.
The term "enzymatic sequencing," "Sanger Method," "dideoxy
technique," and "chain terminator technique," are used
interchangeably herein to describe a method of sequencing DNA named
after its main developer, F. Sanger. The technique uses a
single-stranded DNA template, a short DNA primer and a polymerase
enzyme to synthesize a complementary DNA strand. The primer is
first annealed to the single-stranded template and the reaction
mixture is then split into four aliquots and deoxynucleoside
triphosphates (dNTPs) plus a dideoxynucleoside triphosphate (ddNTP)
are added such that each tube has a different ddNTP. The polymerase
will incorporate a ddNTP opposite its complementary base on the
template but no further dNTPs can be added as the ddNTP lacks a 3'
hydroxyl group. The ratio of ddNTP to dNTP is such that the
polymerase will terminate the growing DNA chain at all positions at
which the ddNTP can be inserted and so a nested set of fragments
(i.e. primer extension products) is formed which all have one end,
the primer, in common. The fragments are labeled so that when the
four reaction mixtures are electrophoresed through a polyacrylamide
gel, a gel band pattern or ladder is formed from which the DNA
sequence can be read directly. The process is shown schematically
in FIG. 7.
The term "enzymatically produced" means produced at least in part
as a result of an action of an enzyme, e.g. fragments of
nucleotides are produced when an enzyme catalyzes a reaction
whereby a larger sequences is cleaved into two or more
fragments.
The term "primer" shall mean a polymer sequence which is
complementary and capable of hybridizing to some part of a single
stranded nucleotide sequence being sequenced which primer is used
to initiate DNA synthesis in vitro.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Sets of fluorescent labels, particularly sets of fluorescently
labeled primers, and methods for their use in multi component
analysis applications, particularly nucleic acid enzymatic
sequencing applications, are provided. At least two of the label
members of the set are energy transfer labels having a common donor
and acceptor fluorophore separated by sufficiently different
distances so that the two labels provide distinguishable
fluorescent signals upon excitation at a common wavelength. In
further describing the subject invention, the subject sets will
first be described in greater detail followed by a discussion of
methods for their use in multi component analysis applications.
Before the subject invention is further described, it is to be
understood that the invention is not limited to the particular
embodiments of the invention described below, as variations of the
particular embodiments may be made and still fall within the scope
of the appended claims. It is also to be understood that the
terminology employed is for the purpose of describing particular
embodiments, and is not intended to be limiting. Instead, the scope
of the present invention will be established by the appended
claims.
It must be noted that as used in this specification and the
appended claims, the singular forms "a," "an" and "the" include
plural reference unless the context clearly dictates otherwise.
Unless defined otherwise all technical and scientific terms used
herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs.
The subject sets of fluorescent labels comprise a plurality of
different types of labels, wherein each type of label in a given
set is capable of producing a distinguishable fluorescent signal
from that of the other types of labels in different sets. Labels in
the different sets generate different signals, preferably, though
not necessarily upon excitation at a common excitation wavelength.
For DNA sequencing applications, the subject sets will comprise at
least 2 different types of labels, and may comprise 8 or more
different types of labels, where for many applications the number
of different types of labels in the set will not exceed 6, and will
usually not exceed four, where at least two of the different types
of labels are energy transfer labels sharing a common donor and
acceptor fluorescer, as described in greater detail below. For
other applications, such as fluorescence in situ hybridization
(FISH), substantially more than 8 labels are ideal so that multiple
targets can be analyzed.
The distinguishable signals generated by the "at least two energy
transfer labels" will at least comprise the intensity of emitted
light at one to two wavelengths. Preferably, the distinguishable
signals produced by the "at least two energy transfer labels" will
comprise distinguishable fluorescence emission patterns, which
patterns are generated by plotting the intensity of emitted light
from differently sized fragments at two wavelengths with respect to
time as differently labeled fragments move relative to a detector,
which patterns are known in the art as electropherograms. For
analyses not based on electrophoresis, such as micro-array chip
based assays, different targets tagged with a specific label can be
differentiated from each other by the unique fluorescence patterns.
For example, in one type of label of a set the intensity of emitted
light at a first wavelength may be twice that of the intensity of
emitted light at a second wavelength and in the second label the
magnitude of the intensities of light emitted at the two
wavelengths may be reversed, or light may be emitted at only one
intensity. The different patterns are generated by varying the
distance between the donor and acceptor. These patterns emitted
from each of these labels are thus distinguishable.
The subject sets will comprise a plurality of different types of
fluorescent labels, where at least two of the labels and usually
all of the labels are energy transfer labels which comprise at
least one acceptor fluorophore and at least one donor fluorophore
in energy transfer relationship, where such labels may have more
complex configurations, such as multiple donors and/or multiple
acceptors, e.g. donor 1, acceptor 1 and acceptor 2. Critical to the
subject sets is that at least two of the labels of the sets have
common donor and acceptor fluorophores, where the only difference
between the labels is the distance between these common acceptor
and donor fluorophores. Thus, for sets of labels in which each
label comprises a single donor and a single acceptor, at least one
of the energy transfer labels will have a donor fluorophore and
acceptor fluorophore in energy transfer relationship separated by a
distance x and at least one of the energy transfer labels will
comprise the same donor and acceptor fluorophores in energy
transfer relationship separated by a different distance y, where
the distances x and y are sufficiently different to provide for
distinguishable fluorescence emission patterns upon excitation at a
common wavelength, as described above. In those sets comprising a
third label having the same donor and acceptor fluorophores as the
first and second label, the distance z between the donor and
acceptor fluorophore will be sufficiently different from x and y to
ensure that the third label is capable of providing a
distinguishable fluorescence emission pattern from the first and
second labels. Thus, in a particular set of labels, one may have a
plurality of labels having the same donor and acceptor
fluorophores, where the only difference among the labels is the
distance between the donor and acceptor fluorophores. To ensure
that different types of labels of a set having common donor and
acceptor fluorophores yield distinguishable fluorescence emission
patterns, the distances between the donor and acceptor fluorophores
will differ by at least about 5%, usually by at least about 10% and
more usually by at least about 20% and will generally range from
about from about 4 to 200 .ANG., usually from about 12 to 100 .ANG.
and more usually from about 15 to 80 .ANG., where the minimums in
such distances are determined based on currently available
detection devices and may be reduced as detection technology
becomes more sensitive, therefore more distinct labels can be
generated.
In one preffered embodiment, at least a portion of, up to and
including all of, the labels of the subject sets sill comprise a
donor and acceptor fluorescer component in energy transfer
relationship and covalently bonded to a spacer component, i.e.
energy transfer labels. Thus, one could have a set of a plurality
of labels in which only two of the labels comprise the above
mentioned donor and acceptor fluorescer components and the
remainder of the labels comprise a single fluorescer component.
Preferably, however, all of the labels will comprise a donor and
acceptor fluorescer component. Generally, for one donor and one
acceptor ET systems, if a set comprises n types of energy transfer
labels, the number of different types of acceptor fluorophores
present in the energy transfer labels of the set will not exceed
n-1. Thus, if the number of different types of energy transfer
labels in the set is four, the number of different acceptor
fluorophores in the set will not exceed 3, and will usually not
exceed 2.
In other preferred embodiments, additional combinations of labels
are possible. Thus, in a set of labels, two of the labels could be
energy transfer labels sharing common donor and acceptor
fluorophores separated by different distances and the remaining
labels could be additional energy transfer labels with different
donor and/or acceptor fluorophores, non-energy transfer fluorescent
labels, and the like.
In the energy transfer labels of the subject sets, the spacer
component to which the fluorescer components are covalently bound
will typically be a polymeric chain or other chemical moiety
capable of acting as a spacer for the donor and acceptor
fluorophore components, such as a rigid chemical moiety, such as
chemicals with cyclic ring or chain structures which can separate
the donor and acceptor and which also can be incorporated with an
active group for attaching to the targets to be analyzed, where the
spacer component will generally be a polymeric chain, where the
fluorescer components are covalently bonded through linking groups
to monomeric units of the chain, where these monomeric units of the
chain are separated by a plurality of monomeric units sufficient so
that energy transfer can occur from the donor to acceptor
fluorescer components. The polymeric chains will generally be
either polynucleotides, analogues or mimetics thereof; or peptides,
peptide analogues or mimetics thereof, e.g. peptoids. For
polynucleotides, polynucleotide analogues or mimetics thereof, the
polymeric chain will generally comprise sugar moieties which may or
may not be covalently bonded to a heterocyclic nitrogenous base,
e.g. adenine, guanine, cytosine, thymine, uracil etc., and are
linked by a linking group. The sugar moieties will generally be
five membered rings, e.g. ribose, or six membered rings, e.g.
hexose, with five membered rings such as ribose being preferred. A
number of different sugar linking groups may be employed, where
illustrative linking groups include phosphodiester,
phosphorothioate, methylene(methyl imino)(MMI), methophosphonate,
phosphoramadite, guanidine, and the like. See Matteucci &
Wagner, Nature (1996) Supp 84: 20-22. Peptide, peptide analogues
and mimetics thereof suitable for use as the polymeric spacer
include peptoids as described in WO 91/19735, the disclosure of
which is herein incorporated by reference, where the individual
monomeric units which are joined through amide bonds may or may not
be bonded to a heterocyclic nitrogenous base, e.g. peptide nucleic
acids. See Matteucci & Wagner supra. Generally, the polymeric
spacer components of the subject labels will be peptide nucleic
acid, polysugarphosphate as found in energy transfer cassettes as
described in PCT/US96/13134, the disclosure of which is herein
incorporated by reference, and polynucleotides as described in
PCT/US95/01205, the disclosure of which is herein incorporated by
reference.
Both the donor and acceptor fluorescer components of the subject
labels will be covalently bonded to the spacer component, e.g. the
polymeric spacer chain, through a linking group. The linking group
can be varied widely and is not critical to this invention. The
linking groups may be aliphatic, alicyclic, aromatic or
heterocyclic, or combinations thereof. Functionalities or
heteroatoms which may be present in the linking group include
oxygen, nitrogen, sulfur, or the like, where the heteroatom
functionality which may be present is oxy; oxo, thio, thiono,
amino, amido and the like. Any of a variety of the linking groups
may be employed which do not interfere with the energy transfer and
gel electrophoresis, which may include purines or pyrimidines,
particularly uridine, thymidine, cytosine, where substitution will
be at an annular member, particularly carbon, or a side chain, e.g.
methyl in thymidine. The donor and/or fluorescer component may be
bonded directly to a base or through a linking group of from 1 to
6, more usually from 1 to 3 atoms, particularly carbon atoms. The
linking group may be saturated or unsaturated, usually having not
more than about one site of aliphatic unsaturation.
Though not absolutely necessarily, generally for DNA sequencing
applications at least one of the donor and acceptor fluorescer
components will be linked to a terminus of the polymeric spacer
chain, where usually the donor fluorescer component will be bonded
to the terminus of the chain, and the acceptor fluorescer component
bonded to a monomeric unit internal to the chain. For labels
comprising polynucleotides, analogues or mimetics thereof as the
polymeric chain, the donor fluorescer component will generally be
at the 5' terminus of the polymeric chain and the acceptor
fluorescer component will be bonded to the polymeric chain at a
position 3' position to the 5 ' terminus of the chain. For other
applications, such as FISH, a variety of labeling approaches are
possible.
The donor fluorescer components will generally be compounds which
absorb in the range of about 300 to 900 nm, usually in the range of
about 350 to 800 nm, and are capable of transferring energy to the
acceptor fluorescer component. The donor component will have a
strong molar absorbance co-efficient 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 molecular
weight of the donor component will usually be less than about 2.0
kD, more usually less than about 1.5 kD. A variety of compounds may
be employed as donor fluorescer components, including fluorescein,
phycoerythrin, BODIPY, DAPI, Indo-1, coumarin, dansyl, cyanine
dyes, and the like. Specific donor compounds of interest include
fluoroscein, rhodamine, cyanine dyes and the like.
Although the donor and acceptor fluorescer component may be the
same, e.g. both may be FAM, where they are different the acceptor
fluorescer moiety will generally absorb light at a wavelength which
is usually at least 10 nm higher, more usually at least 20 nm or
higher, than the maximum absorbance wavelength of the donor, and
will have a fluorescence emission maximum at a wavelength ranging
from about 400 to 900 nm. As with the donor component, the acceptor
fluorescer component will have a molecular weight of less than
about 2.0 kD, usually less than about 1.5 kD. Acceptor fluorescer
moieties may be rhodamines, fluoroscein derivatives. BODIPY and
cyanine dyes and the like. Specific acceptor fluorescer moieties
include FAM, JOE, TAM), ROX, BODIPY and cyanine dyes.
The distance between the donor and acceptor fluorescer components
will be chosen to provide for energy transfer from the donor to
acceptor fluorescer, where the efficiency, of energy transfer will
be from 20 to 100%. Depending on the donor and acceptor fluorescer
components, the distance between the two will generally range from
4 to 200 .ANG., usually from 12 to 100 .ANG. and more usually from
15 to 80 .ANG., as described above.
For the most part the labels of the subject sets will be described
by the following formula: ##STR1## wherein:
D is the donor fluorescer component, which may consist of more than
two different donors separated by a spacer;
N is the spacer component, which may be a polymeric chain or rigid
chemical moiety, where when N is a polymeric spacer that comprises
nucleotides, analogues or mimetics thereof, the number of monomeric
units in N will generally range from about 1 to 50, usually from
about 4 to 20 and more usually from about 4 to 16;
A is the acceptor fluorescer component, which may consist of more
than two different acceptors separated by a spacer; and
X is optional and is generally present when the labels are
incorporated into oligonucleotide primers, where X is a
functionality, e.g. an activated phosphate group, for linking to a
mono- or polynucleotide, analogue or mimetic thereof, particularly
a deoxyribonucleotide, generally of from 1 to 50, more usually from
1 to 25 nucleotides.
For sets to be employed in nucleic acid enzymatic sequencing in
which the labels are to be employed as primers, the labels of the
subject sets will comprise either the donor and acceptor fluorescer
components attached directly to a hybridizing polymeric backbone,
e.g. a polynucleotide, peptide nucleic acid and the like, or the
donor and acceptor fluorescent components will be present in an
energy transfer cassette attached to a hybridizable component,
where the energy transfer cassette comprises the fluorescer
components attached to a non-hybridizing polymeric backbone, e.g. a
universal spacer. See PCT/US96/13134 and ju et al. Nat. ited.
(1996) supra, the disclosures of which are herein incorporated by
reference. The hybridizable component will typically comprise from
about 8 to 40, more usually from about 8 to 25 nucleotides, where
the hybridizable component will generally be complementary to
various commercially available vector sequences such that during
use, synthesis proceeds from the vector into the cloned sequence.
The vectors may include single-stranded filamentous bacteriophage
vectors, the bacteriophage lambda vector, pUC vectors, pGEM
vectors, or the like. Conveniently, the primer may be derived from
a universal primer, such as pUC/M 13, .lambda.gt10, .lambda.gt11,
and the like, (See Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., CSHL, 1989, Section 13), where the universal
primer will have been modified as described above, e.g. by either
directly attaching the donor and acceptor fluorescer components to
bases of the primer or by attaching an energy transfer cassette
comprising the fluorescer components to the primer.
Sets of preferred energy transfer labels comprising donor and
acceptor fluorescers covalently attached to a polynucleotide
backbone in the above D-N-A format include: (1) F6R, F13R, F16R and
F16F; where different formats can employed as long as the four
primers display distinct fluorescence emission patterns.
The fluorescent labels of the subject sets can be readily
synthesized according to known methods, where the subject labels
will generally be synthesized by oligomerizing monomeric units of
the polymeric chain of the label, where certain of the monomeric
units will be covalently attached to a fluorescer component.
The subject sets of fluorescent labels find use in applications
where at least two components of a sample or mixture of components
are to be distinguishably detected. In such applications, the set
will be combined with the sample comprising the to be detected
components under conditions in which at least two of the components
of the sample if present at all will be labeled with first and
second labels of the set, where the first and second labels of the
set comprise the same donor and acceptor fluorescer components
which are separated by different distances. Thus, a first component
of the sample is labeled with a first label of the set comprising
donor and acceptor fluorescer components separated by a first
distance X. A second component of the sample is labeled with a
second label comprising the same donor and fluorescer components
separated by a second distance Y, where X and Y are as described
above. The labeled first and second components, which may or may
not have been separated from the remaining components of the
sample, are then irradiated by light at a wavelength capable of a
being absorbed by the donor fluorescer components, generally at a
wavelength which is maximally absorbed by the donor fluorescer
components. Irradiation of the labeled components results in the
generation of distinguishable fluorescence emission patterns from
the labeled components, a first fluorescence emission pattern
generated by the first label and second pattern being attributable
to the second label. The distinguishable fluorescence emission
patterns are then detected. Applications in which the subject
labels find use include a variety of multicomponent analysis
applications in which fluorescent labels are employed, including
FISH, micro-array chip based assays where the labels may be used as
probes which specifically bind to target components, DNA sequencing
where the labels may be present as primers, and the like.
The subject sets of labels find particular use in polynucleotide
enzymatic sequencing applications, where four different sets of
differently sized polynucleotide fragments terminating at a
different base are generated (with the members of each set
terminating at the same base) and one wishes to distinguish the
sets of fragments from each other. In such applications, the sets
will generally comprise four different labels which are capable of
acting as primers for enzymatic extension, where at least two of
the labels will be energy transfer labels comprising differently
spaced common donor and acceptor fluorescer components that are
capable of generating distinguishable fluorescence emission
patterns upon excitation at a common wavelength of light. Using
methods known in the art, a first set of primer extension products
all ending in A will be generated by using a first of the labels of
the set as a primer. Second, third and fourth sets of primer
extension products terminating in G, C and T will be also be
enzymatically produced. The tour different sets of primer extension
products will then be combined and size separated, usually in an
electrophoretic medium. The separated fragments will then be moved
relative to a detector (where usually either the fragments or the
detector will be stationary). The intensity of emitted light from
each labeled fragment as it passes relative to the detector will be
plotted as a function of time, i.e. an electropherogram will be
produced. Since, the labels of the subject sets will generally emit
light in only two wavelengths, the plotted electropherogram will
comprise light emitted in two wavelengths. Each peak in the
electropherogram will correspond to a particular type of primer
extension product (i.e. A, G, C or T), where each peak will
comprise one of four different fluorescence emission patterns. To
determine the DNA sequence, the electropherogram will be read, with
each different fluorescence emission pattern related to one of the
four different bases in the DNA chain.
Where desired, two sets of labels according to the subject
invention may be employed, where the distinguishable fluorescence
emission patterns produced by the labels in the first set will
comprise emissions at a first and second wavelength and the
patterns produced by the second set of labels will comprise
emissions at a third and fourth wavelength. By using two such sets
in conjunction with one another, one could detect primer extension
products produced from two different template DNA strands at
essentially the same time in a conventional four color detector,
thereby doubling the throughput of the detector.
The subject sets of labels may be sold in kits, where the kits may
or may not comprise additional reagents or components necessary for
the particular application in which the label set is to be
employed. Thus, for sequencing applications, the subject sets may
be sold in a kit which further comprises one or more of the
additional requisite sequencing reagents, such as polymerase,
nucleotides, dideoxynucleotides and the like.
The following examples are offered by way of illustration and not
by way of limitation. The following examples are put forth so as to
provide those of ordinary skill in the art with a complete
disclosure and description of how to make and use the subject sets
of fluorescent labels.
EXPERIMENTAL
A. Design and Synthesis of the Fluorescent Primers
An example of a general labeling scheme using the energy transfer
concept to generate at least eight fluorescent primers from four
fluorescent dyes is described in FIG. 1. To demonstrate the
practicality of the labeling approach, two fluorescent dyes
6-carboxyfluorescein (FAM, F .lambda..sub.em(max) =525 nm) as a
donor and 6-carboxy-X-rhodamine (ROX, R .lambda..sub.em(max) =605
nm, red) as an acceptor are chosen to generate four fluorescent
oligonucleotide primers, which are subsequently used for DNA
sequencing on a cDNA clone. The structures of the fluorescent
primers are presented in FIG. 2. Oligodeoxynucleotides (25-bases
long) with the sequence 5'-TTTTTTTTTTTTTTTTTTTTTTTAC-3'(SEQ ID
NO:01)were synthesized with donor-acceptor fluorophore pairs
separated by different distances. The 25-mer contains a modified
base introduced by the use of
5'-dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acryli
mido]-2'-deoxyuridine,
3'-[(2-cyano-ethyl)-(N,N-diisopropyl)]-phosphoramidite
(Amino-Modifier C6 dT, Glen Research, Sterling, Va.) which has a
protected primary 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. The primers are
synthesized and purified according to the published procedure (Ju,
J., Ruan, C., Fuller, C. W. Glazer, A. N. and Mathies, R. A. (1995)
Proc. Natl. Acad. Sci. USA 92, 4347-4351). 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 intervening nucleotides between D
and A. In all the primers prepared, 6-carboxyfluorescein (FAM, F,
with fluorescence emission maximum at 525 nm) is selected as a
common donor, and 6-carboxy-X-rhodamine (ROX, R, with fluorescence
emission maximum at 605 nm) is selected as an acceptor, except in
one example where N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA,
T, with fluorescence emission maximum at 580 nm) is chosen as an
acceptor as shown in FIG. 3.
Five fluorescent primers with their unique fluorescence signal
patterns are shown in FIG. 3. For primer F6R. the energy transfer
efficiency from donor F to R is higher than 90%, therefore it only
displays a dominant red color from acceptor R. For primer F13R, the
ET efficiency is less than that in F6R, therefore F13R not only
displays a high red signal from the acceptor R and also a blue
signal from F with intensity approximately 40% of the red signal.
For primer F16R, the ET efficiency is even less than that in F13R,
which results in an approximately equal signal intensity from R
(red) and F (blue). For primer F16F carrying two FAM molecules, the
fluorescence signal is dominated by blue. For primer F 16T that
uses TAMRA (T) as an acceptor, the fluorescence signal from F
(blue) is almost in equal intensity as T (Black). It is clear from
these examples that at least five different fluorescent labels are
generated using only three dyes. With two dyes FAM and ROX, four
fluorescent primers are generated that have sufficiently different
fluorescence signals to code for the for DNA sequencing fragments
ended with nucleotides T (F6R), G (F13R, A (F16R) and C (F16F).
These four primers are then chosen for evaluation in DNA sequencing
using a cDNA clone with a polyA tail which can be primed by the
designed primers.
B. DNA Sequencing procedure
Sequencing was performed using a cDNA clone labeled Incyte clone 1
shown below and Thermo Sequenase sequencing kit (Amersham Life
Science) on an ABI 377 sequencer.
Incyte Clone 1
(The italic sequence is the one shown in FIG. 6.) CNCGNCCAGT
GAATTGTAAT ACGACTCACT ATAGGGCGAA TTGGGTACCG (SEQ ID NO:02)
GGCCCCCCCT CGAGTTTTTT TTTTTTTTTT TTTACATGAA GGCAATTTAT TAACAGAAAA
TATTTTGAGG AATCTTGTTC ACAGACGGCG ACCACGGCGA CCCCCCTTCC TGCGAGTGCT
GTCAGAGGGG ATGGGGGTGA CATCCTCAAT CCGCCCGATC ATCATACCCG AGCGGGCAAG
GGCTCTGAGG GCCGACTGGG CCCCAGGTCC AGGGGTCTTG GTCCTATTTC CTCCTGTGGC
CCGGAGTTTG
Four reactions were run, one for each dye/ddNTP combination with
0.2 pmole of the appropriate primer. The reactions containing ddCTP
were run with the F16F primer, ddATP with the F16R primer, ddGTP
with the F13R, and ddTTP with the F6R primer. Fifteen cycles of
94.degree. C. for 20 seconds, 47.degree. C. for 40 seconds and
68.degree. C. for 60 sec were carried out for the sequencing
reaction mixture and then cooled to 4.degree. C. The four reaction
mixtures for each sequence were then combined into one vial and 50
.mu.l of 100% ethanol were added to precipitate the DNA fragments.
The DNA was precipitated by centrifugation for 30 min at 4.degree.
C. and then washed once with 70% ethanol. The precipitated DNA was
vacuum dried, and resuspended in 4 .mu.l of deionized formamide
containing 8.3 mM EDTA and heated at 95.degree. C. for 2 min. The
denatured DNA was loaded on a 4% polyacrylamide 7 M urea denaturing
gel mounted in the instrument. Electrophoresis was conducted for
3.5 hours using 1X Tris-borate-EDTA buffer.
C. DNA Sequencing Results with the Four Fluorescent Primers
FIG. 4 shows that the fluorescence intensity of the single base
extension fragments from primer F6R (T fragments) and F13R (G
fragments) due to energy transfer from F to R is much higher than
that of the single base fragments generated with primer R15R (T
fragments) which carries two ROX dyes but with same sequence as F6R
and F13R. The same concentration of the primer and other sequencing
reagents were used in the comparison. A small portion of the DNA
sequencing raw data in a two color mode sampled from FAM and ROX
using primer F6R, F13R , F16R and F16F on an ABI 377 DNA sequencer
is shown in FIG. 5. From this raw data, sequences can be determined
by the color ratio of the peak in the electropherograms. FIG. 6
shows a large portion of the raw sequencing data (from nucleotide
30 to 130) in 2-color mode generated by primer F6R (T), F13R (G),
F16R (A), F16F (C) and a cDNA clone which has a polyA tail at the
3' end. Sequence can be called by the color patterns of each peak
without applying any mobility shift correction on the raw data. For
example, when the blue and red signals under one peak have almost
the same intensity, the peak is assigned as an A; when only a
dominant blue signal is seen in a peak, it was assigned as a C;
when red signal is slightly higher than the blue signal in a peak,
it was assigned as a G; when the red signal is much higher than the
blue signal in a peak, it was assigned as a T.
It is clear from the experimental data that with two fluorescent
dyes, using energy transfer concepts which offer higher
fluorescence signals, four fluorescent primers can be generated
with sufficiently different fluorescence signal patterns for
sequencing DNA successfully. With two additional different
fluorescent molecules, using the same principle presented, another
four fluorescent primers can be constructed. Thus, with a DNA
sequencer equipped with the appropriate 4-color filters, two sets
of DNA sequencing samples can be analyzed simultaneously, doubling
the sequencing throughput. These sets of unique fluorescent labels
constructed with the concepts presented will find wide applications
in other multiple component analysis projects.
It is evident from the above results and discussion that the sets
of a labels of the subject invention provide for a number of
advantages. For example, where the sets of labels are employed in
DNA sequencing, one can employ a detector capable of detecting at
only two very well separated wavelengths. Therefore, the detector
can sample a large portion of the fluorescence signals, providing
higher sensitivity for detection and increasing readlength of the
sequence. Alternatively, with conventional detectors comprising
four different wavelength detectors, one can effectively double the
throughput obtainable with these detectors by sequencing two
different strands with two sets of labels according to the subject
invention. Thus, the subject sets of labels represent a significant
contribution to the art.
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. The
citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the
present invention is not entitled to antedate such publication by
virtue of prior invention.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it is 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.
* * * * *