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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 methods 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.
U.S.A. (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) =605 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 & 6B shows a 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 (Amersham 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 accceptor 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 will 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 polyncucleotides, 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 fluorescer
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. Med.
(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/M13,
.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 four 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. U.S.A. 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 F16T 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 sqeuencing 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
GGCCCCCCCT
CGAGTTTTTT
TTTTTTTTTT
TTTACATGAA
GGCAATTTAT
TAACAGAAAA
TATTTTGAGG
AATCTTGTTC
ACAGACGGCG
ACCACGGCGA
CCCCCCTTCC
TGCGAGTGCT
GTCAGAGGGG
ATGGGGGTGA
CATCCTCAAT
CCGCCCGATC
TTCATACCCG
AGCGGGCAAG
GGCTCTGAGG
GCCGACTGGG
CCCCAGGTCC
AGGGGTCTTG
GTCCTATTTC
CTCCTGTGGC
CCGGAGTTTG
(SEQ ID NO: 02)
__________________________________________________________________________
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 seconds 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 7M urea denaturing gel
mounted in the instrument. Electrophoresis was conducted for 3.5 hours
using 1.times. 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. Sequences 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 | | |
