U.S. patent application number 10/217615 was filed with the patent office on 2003-01-02 for method, apparatus and kits for sequencing of nucleic acids using multiple dyes.
Invention is credited to Digby, Thomas J., Izmailov, Alexandre.
Application Number | 20030003498 10/217615 |
Document ID | / |
Family ID | 24543162 |
Filed Date | 2003-01-02 |
United States Patent
Application |
20030003498 |
Kind Code |
A1 |
Digby, Thomas J. ; et
al. |
January 2, 2003 |
Method, apparatus and kits for sequencing of nucleic acids using
multiple dyes
Abstract
An instrument for sequencing oligonucleotides is loaded with the
products of four sequencing reaction mixtures. These products are a
combination of A, C, G and T reaction products for several
sequencing reactions. The products of the different sequencing
reactions are labeled with fluorescent tags which are
distinguishable one from the other on the basis of their excitation
or emission spectra. After separation of the oligonucleotides by
electrophoresis, the order of the detected peaks is used to call
the base sequence.
Inventors: |
Digby, Thomas J.; (Toronto,
CA) ; Izmailov, Alexandre; (Toronto, CA) |
Correspondence
Address: |
OPPEDAHL AND LARSON LLP
P O BOX 5068
DILLON
CO
80435-5068
US
|
Family ID: |
24543162 |
Appl. No.: |
10/217615 |
Filed: |
August 12, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10217615 |
Aug 12, 2002 |
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08634284 |
Apr 18, 1996 |
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6432634 |
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10217615 |
Aug 12, 2002 |
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09213834 |
Dec 17, 1998 |
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Current U.S.
Class: |
435/6.12 ;
435/287.2; 536/24.3; 536/24.33 |
Current CPC
Class: |
C12Q 2565/102 20130101;
C12Q 1/6869 20130101; C12Q 1/6869 20130101; C12Q 2531/143 20130101;
C12Q 2537/143 20130101; C12Q 2565/125 20130101; C12Q 2537/143
20130101; C12Q 2535/101 20130101; C12Q 2565/125 20130101; C12Q
2563/107 20130101; C12Q 2565/102 20130101; C12Q 1/6869 20130101;
C12N 15/70 20130101; C12Q 1/6869 20130101; C12N 15/66 20130101;
C12N 15/1034 20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 536/24.3; 536/24.33 |
International
Class: |
C12Q 001/68; C07H
021/04; C12M 001/34; C12M 003/00 |
Claims
What is claimed is:
1. A method for evaluating the nucleic acid sequence of a plurality
of samples comprising the steps of (a) obtaining a first aliquot of
each sample; (b) combining the first aliquot of each sample with a
sequencing reaction mixture containing a polymerase enzyme, a
primer for hybridizing with the sample, nucleoside feedstocks and a
first dideoxynucleoside to form a first plurality of mixtures of
product oligonucleotide fragments, one for each sample, wherein the
product oligonucleotide fragments formed from each first aliquot
are labeled with different fluorescent tags, said different
fluorescent tags being distinguishable one from the other on the
basis of their excitation or emission spectra; (c) combining the
first plurality of mixtures of oligonucleotide products to form a
first combined mixture; (d) loading the first combined mixture onto
a separation matrix at a first loading site; (e) applying an
electric field to cause the oligonucleotide products to migrate
within the separation matrix; and (f) detecting the oligonucleotide
products having the different fluorescent tags as they migrate
within the separation matrix.
2. The method according to claim 1, further comprising the steps
(i) obtaining a second aliquot of each sample; (ii) combining the
second aliquot of each sample with a sequencing reaction mixture
containing a polymerase enzyme, a primer for hybridizing with the
sample, nucleoside feedstocks and a second dideoxynucleoside
different from the first dideoxynucleoside to form a second
plurality of mixtures of product oligonucleotide fragments, one for
each sample, wherein the product oligonucleotide fragments formed
form each second aliquot are labeled with different fluorescent
tags, said different fluorescent tags being distinguishable one
from the other on the basis of their excitation or emission
spectra; (iii) combining the second plurality of mixtures of
oligonucleotide products to form a second combined mixture; (iv)
loading the second combined mixture onto a separation matrix at a
second loading site, distinct from the first loading site; (v)
applying an electric field to cause the oligonucleotide products to
migrate within the separation matrix; and (vi) detecting the
oligonucleotide products having the different fluorescent tags as
they migrate within the separation matrix.
3. The method according to claim 2, wherein the first and second
aliquots are combined with a sequencing reaction concurrently, and
wherein the first and second combined mixtures are loaded onto
different lanes of the same gel.
4. The method according to claim 3, wherein the same primer and
fluorescent tag are combined with the first and second aliquots of
each sample.
5. The method according to claim 1, wherein first aliquots from at
least three samples are combined to form the first combined
mixture.
6. The method according to claim 1, wherein first aliquots from
more than four samples are combined to form the first combined
mixture.
7. A method for evaluating the sequence of a target nucleic acid
polymer in a plurality of samples comprising the steps of (a)
obtaining four aliquots of each sample; (b) combining the aliquots
of each sample with four sequencing reaction mixtures, each
sequencing reaction mixture containing a polymerase enzyme, a
primer for hybridizing with the target nucleic acid, nucleoside
feedstocks and a different dideoxynucleoside to form an A-mixture,
a G-mixture, a T-mixture and a C-mixture for each sample containing
product oligonucleotide fragments of varying lengths, wherein the
product oligonucleotide fragments are labeled with fluorescent
tags, and the fluorescent tags used for each sample are
distinguishable one from the other on the basis of their excitation
or emission spectra; (c) combining the A-mixtures, the C-mixtures,
the T-mixtures and the C-mixtures for each sample to form a
combined A-mixture, a combined C-mixture, a combined t-mixture and
a combined C-mixture; (d) loading the combined A-mixture, the
combined G-mixture, the combined T-mixture and the combined
C-mixture onto a separation matrix at separate loading sites; (e)
applying an electric field to cause the product oligonucleotide
fragments to migrate within the separation matrix; and (f)
detecting the product oligonucleotide fragments having the
different fluorescent tags as they migrate within the separation
matrix.
8. An apparatus for evaluating the sequence of a nucleic acid
polymer by separation of a reaction mixture containing
oligonucleotide fragments in a separation matrix disposed within
the apparatus comprising (a) means for providing excitation energy
to a detection site within the separation matrix; (b) means for
detecting light emitted from fluorescently-labeled oligonucleotide
fragments located within the detection zone; (c) configuration
control means, operatively connected to the means for providing
excitation energy and the means for detecting to provide
combinations of excitation wavelength and detection wavelength
specific for a plurality of different fluorescently-labeled
oligonucleotide fragments; and (d) data processing means,
operatively connected to the configuration control means and the
means for detecting for receiving a signal from the means for
detecting and assigning that signal to a data stream based upon the
combination of excitation wavelength and detection wavelength set
by the configuration control means.
9. A kit for determining the sequence of a selected region of DNA
comprising, in packaged combination, at least of first set of a
plurality of containers, each container containing a reagent
species for the sequencing of the selected region of DNA, wherein
the reagent species in each container comprises a reactive portion
and a label portion and wherein the label portions of the reagents
are different and distinguishable one from the other.
10. The kit according to claim 9, wherein the detectable labels are
fluorescent tags, distinguishable on the basis of the excitation or
emission spectra thereof.
11. The kit according to claim 10, wherein the reactive portion of
each reagent species in the first set of containers is an
oligonucleotide primer for use in sequencing the selected region of
DNA.
12. The kit according to claim 11, wherein the reactive portions of
each reagent species in the first set of containers are the same as
one another.
13. The kit according to claim 11, wherein the reactive portion of
each reagent species in the first set of containers is a
dideoxynucleoside and wherein the reactive portions of each reagent
species in the first set of containers are the same as one
another.
14. The kit according to claim 13, further comprising a second set
of containers, each containing a reagent species for the sequencing
of the selected region of DNA, wherein the reagent species in each
container of the second set comprises a dideoxynucleoside reactive
portion different from the reactive portion of the reagent species
in the first set of containers, and a fluorescent label portion and
wherein the label portions of the reagents in the second set of
containers are different and distinguishable one from the other on
the basis of the excitation or emission spectra thereof.
15. A method for evaluating the sequence of a plurality of gene
regions within a sample comprising the steps of: (a) combining at
least a first aliquot of the sample with a sequencing reaction
mixture containing a polymerase enzyme, a plurality of sequencing
primer species, one sequencing primer species for each gene region,
nucleoside feedstocks and a first dideoxynucleoside to form a first
mixture of product oligonucleotide fragments, wherein each of the
sequencing primer species is labeled with a different detectable
label, said different detectable labels being distinguishable one
from the other by a detection system; (b) separating the first
mixture of product oligonucleotide fragments based upon the size of
the fragments; (c) detecting emissions from the separated
oligonucleotide fragments for each different detectable label; and
(d) evaluating the sequence of each gene region based upon the
oligonucleotide fragments detected.
16. The method according to claim 15, wherein the detectable labels
are fluorescent tags.
17. The method according to claim 16, wherein fluorescent tags are
distinguishable based upon their excitation or emission
spectra.
18. The method according to claim 15, wherein four aliquots of
sample are combined with four separate sequencing reaction
mixtures, each containing a different dideoxynucleoside.
19. A kit for evaluating the sequence of a plurality of gene
regions within a sample comprising, in packaged combination, at
least one container containing a mixture of a plurality of
sequencing primers, one for each gene region to be evaluated,
wherein the plurality of sequencing primers each comprise a
reactive portion which hybridizes with DNA in the sample and a
label portion and wherein the label portions of the reagents are
different and distinguishable one from the other.
20. The kit according to claim 20, wherein the detectable labels
are fluorescent tags, distinguishable one from the other by their
emission or excitation spectra.
Description
BACKGROUND OF THE INVENTION
[0001] This application relates to an improved method for
sequencing of nucleic acids using multiple fluorescent labels, and
to apparatus and kits adapted for use with the method.
[0002] Sequencing of nucleic acids using the chain termination
method involves the general steps of combining the target nucleic
acid polymer to be sequenced with a sequencing primer which
hybridizes with the target nucleic acid polymer; extending the
sequencing primer in the presence of normal nucleotide (A, C, G,
and T) and a chain-terminating nucleotide, such as a
dideoxynucleotide, which prevents further extension of the primer
once incorporated; and analyzing the product for the length of the
extended fragments obtained. Analysis of fragments may be done by
electrophoresis, for example on a polyacrylamide gel.
[0003] Although this type of analysis was originally performed
using radiolabeled fragments which were detected by autoradiography
after separation, modern automated DNA sequencers generally are
designed for use with sequencing fragments having a fluorescent
label. The fluorescently labeled fragments are detected in real
time as they migrate past a detector.
[0004] U.S. Pat. No. 5,171,534 which is incorporated herein by
reference describes a variation of this basic sequencing procedure
in which four different fluorescent labels are employed, one for
each sequencing reaction. The fragments developed in the A, G, C
and T sequencing reactions are then recombined and introduced
together onto a separation matrix. A system of optical filters is
used to individually detect the fluorophores as they pass the
detector. This allows the throughput of a sequencing apparatus to
be increased by a factor of four, since the four sequencing
reaction which were previously run in four separate lanes or
capillaries can now be run in one.
[0005] It is an object of the present invention to provide a
further improvement for use with chain termination sequencing
reactions which can further increase the throughput of an
instrument.
SUMMARY OF THE INVENTION
[0006] In order to use nucleic acid sequencing as a diagnostic
tool, it will be necessary to determine the sequence of the same
DNA region from many samples. The present invention makes it
possible to increase the throughput of an instrument being used for
this purpose. Thus, a first aspect of the invention provides a
method for evaluating the sequence of a target nucleic acid polymer
in a plurality of samples. In this method, each sample is first
divided into four aliquots which are combined with four sequencing
reaction mixtures. Each sequencing reaction mixture contains a
polymerase enzyme, a primer for hybridizing with the target nucleic
acid, nucleoside feedstocks and a different dideoxynucleoside. This
results in the formation of an A-mixture, a C-mixture, a T-mixture
and a C-mixture for each sample containing product oligonucleotide
fragments of varying lengths. The product oligonucleotide fragments
are labeled with fluorescent tags, and these tags will generally be
the same for all four sequencing reactions for a sample. However,
the fluorescent tags used for each sample are distinguishable one
from the other on the basis of their excitation or emission
spectra.
[0007] Next, the A-mixtures for each sample are combined to form a
combined A mixture, the C-mixtures are combined to form a combined
G-mixture and so on for all four mixtures. The combined mixtures
are loaded onto a separation matrix at separate loading sites and
an electric field is applied to cause the product oligonucleotide
fragments to migrate within the separation matrix. The separated
product oligonucleotide fragments having the different fluorescent
tags are detected as they migrate within the separation matrix.
[0008] The method of the invention can be used as described above
to determine the position of every base in the sequence, or it can
be used to determine the position of less than all four bases. For
example, the method can be used to determine the position of only
the A bases within a sequence for some diagnostic applications.
[0009] A further aspect of the present invention is a kit useful
for diagnostic sequencing of a selected portion of a gene. One
embodiment of such a kit contains a plurality of sequencing primers
for the selected portion of the gene, each sequencing primer being
identical in its DNA sequence but being labeled with a different
fluorescent tag.
[0010] A further aspect of the invention is an apparatus for
performing the method of the invention. Such an apparatus
comprises
[0011] (a) means for providing excitation energy to a detection
site within a separation matrix disposed within the apparatus;
[0012] (b) means for detecting light emitted from
fluorescently-labeled oligonucleotide fragments located within the
detection site;
[0013] (c) configuration control means, operatively connected to
the means for providing excitation energy and the means for
detecting to provide combinations of excitation wavelength and
detection wavelength specific for a plurality of different
fluorescently-labeled oligonucleotide fragments; and
[0014] (d) data processing means, operatively connected to the
configuration control means and the means for detecting for
receiving a signal from the means for detecting and assigning that
signal to a data stream based upon the combination of excitation
wavelength and detection wavelength set by the configuration
control means.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A and B shows a schematic representation of the
method of the invention;
[0016] FIGS. 2A, B, C, and D show excitation and emission spectra
for theoretical sets of useful fluorescent tags; and
[0017] FIG. 3 shows an apparatus for evaluating the sequence of
nucleic acid polymers using the method of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0018] FIG. 1A shows a schematic representation of one embodiment
of the method of the invention. The figure depicts the application
of the method to two samples for clarity. As will be apparent from
the discussion below, however, the method of the invention is not
limited to two samples, and is in fact preferably applied for four
or more samples, up to a limit imposed only by the number of
distinguishable tags which can be identified.
[0019] As shown in FIG. 1A, two samples, "sample 1" and "sample 2"
are each divided into four aliquots and these aliquots are
introduced into sequencing reactions A1, C1, G1, and T1, and A2,
C2, G2 and T2. Each sequencing reaction contains the reagents
necessary for producing product oligonucleotide fragments of
varying lengths indicative of the position of one-base within the
target nucleic acid sequence. These reagents include a polymerase
enzyme, for example T7 polymerase, Sequenase.TM., Thermo
Sequenase.TM., or the Klenow fragment of DNA polymerase; A, C, G
and T nucleoside feedstocks; one type of chain terminating
dideoxynucleoside; and a sequencing primer.
[0020] After the product oligonucleotide fragments are formed in
each reaction mixture, the products from reaction mixture A1 are
combined with the products from reaction mixture A2 to form a
combined mixture 10 which is loaded onto lane 1 of a separation
matrix. Likewise, the products from reaction mixture C1 are
combined with the products from reaction mixture C2 to form a
combined mixture 11 which is loaded onto lane 2 of the separation
matrix; the products from reaction mixture C1 are combined with the
products from reaction mixture G2 to form a combined mixture 12
which is loaded onto lane 3 of the separation matrix; and the
products from reaction mixture T1 are combined with the products
from reaction mixture T2 to form a combined mixture 13 which is
loaded onto lane 4 of the separation matrix.
[0021] The key to the present invention is the use of labels in the
reactions A1, C1, G1, and T1 which are distinguishable from the
labels used in reactions A2, C2, C2 and T2, respectively. Thus,
unlike the method described in U.S. Pat. No. 5,171,534 where the
labels used for the A, C, C, and T reactions for a sample are
distinct, in the present invention the labels used for the four
sequencing reactions for any one sample can be, and preferably are,
the same. Instead, it is the labels which are used in the several
samples which are distinct in the method of the invention.
[0022] An alternative embodiment of the invention is illustrated in
FIG. 1B. In this case, the operator wants to sequence a plurality
of genes (or different exons of the same gene) from one patient
sample. The sample 20 is divided into four aliquots. A sequencing
reaction mix containing the reagents necessary for producing
product oligonucleotide fragments of varying lengths is added to
each aliquot. The sequencing mix added to a first aliquot contains
all of the reagents for an A termination reaction, plus a plurality
of sequencing primers, each one labeled with a distinguishable
fluorophore, and each one being specific for a different gene (or
different exon of the same gene). The sequencing mix added to a
second aliquot contains all of the reagents for a C termination
reaction, plus the same plurality of sequencing primers. Sequencing
reaction mixes for G and T are made in the same fashion. These
sequencing mixture are reacted to produce oligonucleotides
fragments, and then loaded onto lanes 21, 22, 23, and 24 of a
sequencing gel and separated. Using this technique, any number of
genes or exons in a sample can be simultaneously sequenced up to
the limit imposed by the number distinguishable tags which can be
identified.
[0023] Suitable labels for use in the present invention are
fluorescent tags. These can be incorporated into the product
oligonucleotide fragments in any way, including the use of
fluorescently tagged primers or fluorescently tagged chain
terminating reagents.
[0024] The fluorescent tags selected for use in the present
invention must be distinguishable one from another based on their
excitation and/or emission spectra. For example, as shown in FIG.
2A, a set of tags could be selected which had overlapping emission
spectra (Em1, Em2, Em3 and Em4) but separate and distinguishable
excitation spectra (Exl, Ex2, Ex3, and Ex4). A set of tags could
also be selected which had overlapping excitation spectra but
separate and distinguishable emission spectra as shown in FIG. 2B.
Further, as shown in FIG. 2C, a set of tags could be selected in
which some of the tags have overlapping excitation spectra (Ex1 and
Ex2) but separate and distinguishable emission spectra (Em1 is
distinguishable from Em2); while the others have separate and
distinguishable excitation spectra (Ex1, Ex3, and Ex4) but
overlapping emission spectra (Em1, Em3 and Em4). A further
combination of excitation and emission spectra is shown in FIG.
2D.
[0025] Examples of sets of suitable tags, together with the
wavelength maximum for the excitation and emission spectra are
shown in Table 1. Many other fluorophores are available that can be
used as labels for DNA sequencing reaction products. Such dyes are
available from Applied Biosystems, Inc. (Foster City, Calif.),
Molecular Probes, Inc. (Oregon) and others.
1TABLE 1 Fluorescent Dye's suitable for use with the invention
Excitation Fluorescent Dye Max (nm) Emission Max (nm) Texas Red X
599 617 Carboxy-X-Rhodamine 585 612 CarboxyFluorescein 494 521
CarboxyTetraMethylRhodamine 561 591 Carboxycyanine 5.0 650 667
[0026] FIG. 3 shows a basic layout for an apparatus for evaluating
the sequence of nucleic acid polymers using the method of the
invention. Light from a light source 31, which may be for example a
laser, a light emitting diode, a laser diode, an incandescent or
polychromatic lamp, or any combination of such sources, is passed
through an optical filter 32 to select an appropriate excitation
wavelength which is directed to a detection site 33 in a separation
matrix 34. Light emitted by fluorescent tags in the detection site
33 passes through a second optical filter 35 to a detector 36.
Either or both of the optical filters 32 and 36 may be adjustable
under the control of a microprocessor, minicomputer or personal
computer 37 to provide various configurations of excitation and
emission wavelengths as discussed more fully below. The output from
the detector is then transmitted to a data processing system such
as a dedicated microprocessor, minicomputer or personal computer 37
for analysis to produce a report on the sequence of the sample
being evaluated.
[0027] In the case where the properties of the selected tags are of
the type shown in FIG. 2A, the optical filter 32 may adjustable,
for example by rotating several different filters through the path
of the excitation beam, to produce excitation beams corresponding
to the different excitation wavelengths of the tags. Optical filter
35 may then be simply a cut-off filter selected to exclude light of
the excitation wavelengths from the detector. Information
concerning the position of the optical filter 32 as a function of
time is transmitted to the data processing system, and used to
permit interpretation of the fluorescence data. Thus, when the
optical filter 32 is in a position that corresponds to the
excitation spectrum of the tag used to label sample 1, the data
processing system interprets the emission intensity as data for the
sequence of sample 1; when the optical filter 32 is in a position
that corresponds to the excitation spectrum of the tag used to
label sample 2, the data processing system interprets the emission
intensity as data for the sequence of sample 2 and so on for as
many different tags are used.
[0028] In the case where the properties of the selected tags are of
the type shown in FIG. 2B, the optical filter 35 is adjustable, for
example by rotating several different filters through the path of
the excitation beam, to selectively collect emissions wavelengths
corresponding to the different tags. Optical filter 32 may be
simply a cut-off or band-pass filter selected to exclude light of
the emission wavelengths from the detector. Information concerning
the position of the optical filter 35 as a function of time is
transmitted to the data processing system, and used to permit
interpretation of the fluorescence data. Thus, when the optical
filter 35 is in a position that corresponds to the emission
spectrum of the tag used to label sample 1, the data processing
system interprets the emission intensity as data for the sequence
of sample 1; when the optical filter 35 is in a position that
corresponds to the emission spectrum of the tag used to label
sample 2, the data processing system interprets the emission
intensity as data for the sequence of sample 2 and so on for as
many different tags are used.
[0029] Finally, in the case where the properties of the selected
tags are of the type shown in FIG. 2C, both optical filter 32 and
optical 35 are adjustable in synchronization to control the
excitation and emission wavelengths being monitored. Information
concerning the position of the optical filters 32 and 35 as a
function of time is transmitted to the data processing system, and
used to permit interpretation of the fluorescence data. Thus, when
optical filter 32 is in a position that corresponds to excitation
spectrum Ex1 in FIG. 2C, and optical filter 35 is in a position
that transmits the light of the wavelength of emission spectrum
Em1, the data processing system interprets the emission intensity
as data for the sequence of the sample labeled with tag 1. When the
optical filter 35 is in a position to transmit light of the
wavelength of emission spectrum Em2, on the other hand, the data
processing system interprets the emission intensity as data for the
sequence of the of sample labeled with tag 2.
[0030] The light source 31 may be a single light source which is
moved across the gel to irradiate each detection zone individually,
for example as described in U.S. Pat. No. 5,207,880 which is
incorporated herein by reference. The light source 31 may also be a
singe light source which is split into multiple beamlets, for
example using optical fibers or through the use of a beam splitter
such as a spot array generation grating to each of the detection
sites as described generally in U.S. patent application Ser. No.
08/353,932 and PCT Patent Application No. PCT/US95/15951 which are
incorporated herein by reference. The light source 31 may also be
multiple individual light sources, each of which irradiates a
subset of one or more of the detection sites within the separation
matrix.
[0031] While optical filter 32 described above can be used
effectively to provide selection of excitation wavelength, it will
be understood that other approaches to providing different
excitation wavelengths can be used as well. For example a plurality
of lasers of different wavelengths could be used, with the light
from each directed in turn to the detection site. For detection of
product oligonucleotide fragments in multiple lanes of an
electrophoresis gel, one set of lasers might be used (one for each
necessary wavelength) with light from each of the lasers being
conducted by optical fibers or through the use of a beam splitter
such as a spot array generation grating to each of the detection
sites as described generally in U.S. patent application Ser. No.
08/353,932 and PCT Patent Application No. PCT/US95/15951. Optical
switches, operatively connected to the data processing system, are
used to select which of the excitation wavelengths is striking the
detection zone at any given time, in the same manner as a rotating
optical filter could do. A detector assembly which scans across the
lanes of an electrophoresis gel could also be used, for example as
described in U.S. Pat. Nos. 5,360,523 and 5,100,529, which are
incorporated herein by reference, although the time required for
such a device to scan all the lanes of a gel may be a limiting
factor in applications with short total migration times.
[0032] It will also be appreciated that multiple optical filters
mounted on individual detectors could be used in place of the
adjustable optical filter 35 and the single detector 36 shown in
FIG. 3. Similarly several detectors with adjustable optical filters
might also be used.
[0033] Other optical components which separate light by wavelength
may also be used in place of optical filter 35. Thus, for example,
a diffraction grating or prism which spatially separates light of
differing wavelength may be employed. In this case, radiation from
the different fluorophores will be distributed in space, and can be
detected by dedicated detectors such as photodiodes, CCD elements
(linear or X-Y). Similarly, distinct optical filters for each
wavelength can be employed in combination with a multiplicity of
detectors. Optical filters which are transmissive at the emission
wavelength of one fluorophore and reflective at the emission
wavelength of a second fluorophore can also be used to direct
emitted light to separate detectors depending on wavelength.
[0034] The detectors used in the apparatus of the invention may be
photomultipliers, photodiodes or a CCD. As noted above, the
apparatus can be configured with one or more detectors for each
detection site. The apparatus can also be configured such that one
detector overlaps with several detection sites if the excitation
light is directed to the detection sites one at a time. In
addition, the apparatus can use a single detector (or a small array
of detectors) which is scanned across the gel during detection.
[0035] For determining the sequence of a selected region of DNA
using the method of the invention, a kit may be formulated. Such a
kit comprises, in packaged combination, a plurality of containers,
each containing a reagent for the sequencing of the selected region
of DNA. The reagent in each container has a reactive portion, which
is involved in the sequencing reaction, and a fluorescent label
portion. The label portions of the reagents in each container are
different and distinguishable one from the other on the basis of
the excitation or emission spectra thereof.
[0036] In a preferred embodiment, a kit in accordance with the
invention comprises a plurality of primers for sequencing the
selected region, each of the primers having a different and
distinguishable fluorescent label. Thus, the reactive portions of
the reagents in this case are the oligonucleotide primer to which
the labels are attached. The reactive portions may be different
from one another, but are preferably the same. Such a kit may also
include additional reagents for sequencing, including polymerase
enzymes, dideoxynucleosides and buffers.
[0037] Alternatively, the kit may contain one primer for the
selected region and a plurality of containers of chain-terminating
nucleosides, each labeled with a different and distinguishable
fluorescent label. In this case, the reactive portion of the
reagent is the chain terminating nucleoside which can be
incorporated in place of a normal nucleoside during the sequencing
reaction. The kit may include reagents having just one type of
chain-terminating nucleoside, for example ddA with a plurality of
distinct fluorescent labels, or it may include reagents having two
or more types of chain-terminating nucleosides, each with a
plurality of distinct labels. Such a kit may also include
additional reagents for sequencing, including polymerase enzymes
and buffers, as well as additional chain-terminating nucleosides
(single-labeled) for those not provided as part of a multi-label
reagent set.
[0038] For practising the method shown in FIG. 1B, a suitable kit
in accordance with the invention includes at least one container
containing a mixture of a plurality of sequencing primers, one for
each gene region to be evaluated. The plurality of sequencing
primers each comprise a reactive portion which hybridizes with DNA
in the sample and a label portion, the label portions of the
reagents being different and distinguishable one from the other.
Preferably, the detectable labels are fluorescent tags,
distinguishable one from the other by their emission or excitation
spectra.
* * * * *