U.S. patent application number 09/776291 was filed with the patent office on 2002-09-05 for automated dna sequencing technique.
Invention is credited to Connell, Charles R., Hood, Leroy E., Hunkapiller, Michael W., Hunkapiller, Tim J., Smith, Lloyd M..
Application Number | 20020123046 09/776291 |
Document ID | / |
Family ID | 46251268 |
Filed Date | 2002-09-05 |
United States Patent
Application |
20020123046 |
Kind Code |
A1 |
Smith, Lloyd M. ; et
al. |
September 5, 2002 |
Automated DNA sequencing technique
Abstract
A process for the electrophoretic analysis of DNA fragments
produced in DNA sequencing operations wherein chromophores or
fluorophores are used to tag the DNA fragments produced by the
sequencing chemistry and permit the detection and characterization
of the fragments as they are resolved by electrophoresis through a
gel. Preferably four different fragment sets are tagged with the
fluorophores fluorescein, Texas Red, tetramethyl rhodamine, and
7-nitro-benzofurazan. A system for the electrophoretic analysis of
DNA fragments produced in DNA sequencing operations comprising: a
source of chromophore or fluorescent tagged DNA fragments; a zone
for contacting an electrophoresis gel; means for introducing said
tagged DNA fragments to said zone; and photometric means for
monitoring said tagged DNA fragments as they move through said
gel.
Inventors: |
Smith, Lloyd M.; (Madison,
WI) ; Hood, Leroy E.; (Seattle, WA) ;
Hunkapiller, Michael W.; (San Carlos, CA) ;
Hunkapiller, Tim J.; (Seattle, WA) ; Connell, Charles
R.; (Redwood City, CA) |
Correspondence
Address: |
Debra J. Glaister
Morrison & Foerster LLP
755 Page Mill Road
Palo Alto
CA
94304-1018
US
|
Family ID: |
46251268 |
Appl. No.: |
09/776291 |
Filed: |
February 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09776291 |
Feb 2, 2001 |
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08484340 |
Jun 7, 1995 |
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6200748 |
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08484340 |
Jun 7, 1995 |
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08361176 |
Dec 21, 1994 |
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5821058 |
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08361176 |
Dec 21, 1994 |
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07898019 |
Jun 12, 1992 |
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07898019 |
Jun 12, 1992 |
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08066327 |
May 21, 1993 |
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08066327 |
May 21, 1993 |
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07660160 |
Feb 21, 1991 |
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07660160 |
Feb 21, 1991 |
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07106232 |
Oct 7, 1987 |
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07660160 |
Feb 21, 1991 |
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07558312 |
Oct 15, 1990 |
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5171534 |
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07558312 |
Oct 15, 1990 |
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06722742 |
Apr 11, 1985 |
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07558312 |
Oct 15, 1990 |
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06689013 |
Jan 2, 1985 |
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06689013 |
Jan 2, 1985 |
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06570973 |
Jan 16, 1984 |
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Current U.S.
Class: |
435/6.11 ;
536/25.32 |
Current CPC
Class: |
G01N 27/44726 20130101;
C12Q 1/6869 20130101; C12Q 2535/101 20130101; C12Q 2563/107
20130101; G01N 27/44721 20130101; C12Q 1/6869 20130101; C12Q 1/6816
20130101 |
Class at
Publication: |
435/6 ;
536/25.32 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
1. A process for the electrophoretic analysis of DNA fragments
produced in DNA sequencing operations which comprises providing
tagged DNA fragments having at least one chromophore or fluorophore
produced by the sequencing chemistry, and detecting said fragments
as they are resolved by electrophoresis through a gel.
2. The method of DNA sequencing by the chain termination method
according to claim 1 wherein a primer oligonucleotide labeled with
a colored tag is used.
3. The method of DNA sequencing by the chain termination method
according to claim 1 wherein a primer oligonucleotide labeled with
a fluorescent tag is used.
4. The method of DNA sequencing by chemical degradation method
according to claim 1 wherein DNA molecules labeled with a colored
tag are used.
5. The method of DNA sequencing by chemical degradation method
according to claim 1 wherein DNA molecules labeled with a
fluorescent tag are used.
6. The method of DNA sequencing according to claim 1 wherein a set
of four chromophores or fluorophores are used to tag said DNA
fragments produced by the sequencing chemistry.
7. In the method of DNA sequencing by the chain termination method;
the improvement wherein the primer oligonucleotide used in each of
the four sequencing reactions, A, C, G and T, has a different
colored tag attached to it, and wherein aliquots of the aforesaid
sequencing reactions are combined and electrophoresed together on
polyacrylamide gel and detected after their separation on the
gel.
8. In the method of DNA sequencing by the chain termination method;
the improvement wherein the primer oligonucleotide used in each of
the four sequencing reactions, A, C, G and T, has a different
fluorescent tag attached to it, and wherein aliquots of the
aforesaid sequencing reactions are combined and electrophoresed
together on polyacrylamide gel and detected after their separation
on the gel.
9. In the method of DNA sequencing by chemical degradation method;
the improvement wherein the DNA molecules are labeled with
different colored tags, and a different colored DNA is used in each
of the chemical modification reactions, and aliquots of the
aforesaid sequencing reactions are combined and electrophoresed
together on a polyacrylamide gel and detected after their
separation of the gel.
10. In the method of DNA sequencing by chemical degradation,
method; the improvement wherein the DNA molecules are labeled with
different fluorescent tags, and a different fluorescent DNA is used
in each of the chemical modification reactions, and aliquots of the
aforesaid sequencing reactions are combined and electrophoresed
together on a polyacrylamide gel and detected after their
separation of the gel.
11. In the method of DNA sequencing by chemical degradation method;
the improvement wherein the DNA molecules are provided with an
amino group, which is coupled to a dye molecule subsequent to the
sequencing reactions.
12. In the method of DNA sequencing by chemical degradation method;
the improvement wherein the DNA molecules are provided with a
protected amino group, which is deblocked and coupled to a dye
molecule subsequent to the sequencing reactions.
13. In the method of claim 11, the further improvement wherein the
products of each of the different sequencing reactions are coupled
with a different color dye, aliquots of the dye labeled reaction
are combined and electrophoresed on a polyacrylamide gel and
detected after their separation on the gel.
14. In the method of claim 12, the further improvement wherein the
products of each of the different sequencing reactions are coupled
with a different color dye, aliquots of the dye labeled reaction
are combined and electrophoresed on a polyacrylamide gel and
detected after their separation on the gel.
15. In the process for the electrophoretic analysis of DNA fragment
sets produced in DNA sequencing operations wherein a set of four
fluorophores are used to tag the DNA fragments produced by the
sequencing chemistry and permit the detection and characterization
of the fragments as they are resolved by electrophoresis through a
gel, the improvement wherein the four different fragment sets are
tagged with the fluorophores fluorescein, Texas Red, tetramethyl
rhodamine, and 7-nitro-benzofurazan.
16. The method of claim 15 wherein the DNA sequencing is carried
out by the chain termination method.
17. In the method of claim 15 where the DNA sequencing is carried
out by the chemical degradation method comprising modification and
cleavage reactions.
18. In the method of claim 17 wherein the DNA fragments are labeled
with dye prior to the modification reactions.
19. In the method of claim 17 wherein the DNA fragments are labeled
with dye subsequent to the modification reactions but prior to the
cleavage reactions.
20. In the method of claim 17 wherein the DNA fragments are labeled
with dye subsequent to the cleavage reactions.
21. In the method of DNA sequencing by the chain termination method
comprising four sequencing reactions; the improvement wherein the
primer oligonucleotides used in the sequencing reactions, A, C, G
and T, has a different fluorescent tag attached to it, and wherein
aliquots of the aforesaid sequencing reactions are combined and
electrophoresed together on a polyacrylamide gel and detected after
their separation on the gel, said fluorescent tags
being-fluorescein, Texas Red, tetramethyl rhodamine, and
7-nitro-benzofurazan.
22. In the method of DNA sequencing by the chemical degradation
method comprising modification and cleavage reactions; the
improvement wherein the DNA molecules are labeled with different
fluorescent tags, and a different fluorescent DNA is used in each
of the chemical modification reactions, and aliquots of the
aforesaid sequencing reactions are combined and electrophoresed
together on a polyacrylamide gel and detected after their
separation of the gel, said fluorescent tags being fluorescein,
Texas Red, tetramethyl rhodamine, and 7-nitro-benzofurazan.
23. A novel system for the electrophoretic analysis of DNA
fragments produced in DNA sequencing operations comprising: a
source of chromophore or fluorescent tagged DNA frag- ments from
sequencing operations, a zone for containing an electrophoresis
gel, means for introducing said tagged DNA fragments to said zone;
and photometric means for monitoring said tagged DNA frag- ments as
they move through said gel.
24. The novel system of claim 23 wherein the photometric means is
an absorption photometer.
25. The novel system of claim 23 wherein the photometric means is
an fluorescent photometer.
26. The novel system of claim 23 wherein the DNA fragments are
labeled with an amino group which is coupled to a dye molecule.
27. The novel system of claim 23 wherein a set of four chromophores
or fluorophores are present to tag said DNA fragments from
sequencing operations.
28. A novel system for the electrophoretic analysis of DNA
fragments produced in DNA sequencing operations comprising: a
source of chromophore or fluorescent tagged DNA fragments from
sequencing operations; a zone containing an electrophoresis gel;
means for introducing said tagged DNA fragments to said zone; and
photometric means for monitoring said tagged DNA fragments as they
move through said gel.
29. The novel system of claim 28 wherein said source of tagged DNA
fragments from sequencing operations is positioned at one end of
said zone, and said detector is positioned in proximity to the
opposite end of said zone.
30. The novel system of claim 28 wherein a set of four chromophores
or fluorophores are present to tag said DNA fragments from
sequencing operations.
Description
[0001] This is a continuation-in-part of U.S. Ser. No. 722,742,
filed Apr. 11, 1985 which was a continuation-in-part of U.S. Ser.
No. 689,013 filed Jan. 2, 1985 which, in turn, was a
continuation-in-part of U.S. Ser. No. 570,973, filed Jan. 16, 1984,
now abandoned.
BACKGROUND OF THE INVENTION
[0002] The development of reliable methods for sequence analysis of
DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) has been one
of the keys to the success of recombinant DNA and genetic
engineering. When used with the other techniques of modern
molecular biology, nucleic acid sequencing allows dissection and
analysis of animal, plant and viral genomes into discrete genes
with defined chemical structure. Since the function of a biological
molecule is determined by its structure, defining the structure of
a gene is crucial to the eventual manipulation of this basic unit
of hereditary information in useful ways. Once genes can be
isolated and characterized, they can be modified to produce desired
changes in their structure that allow the production of gene
products--proteins--with different properties than those possessed
by the original proteins. Microorganisms into which the natural or
synthetic genes are placed can be used as chemical "factories" to
produce large amounts of scarce human proteins such as interferon,
growth hormone, and insulin. Plants can be given the genetic
information to allow them to survive harsh environmental conditions
or produce their own fertilizer.
[0003] The development of modern nucleic acid sequencing methods
involved parallel developments in a variety of techniques. One was
the emergence of simple and reliable methods for cloning small to
medium-sized strands of DNA into bacterial plasmids,
bacteriophages, and small animal viruses. This allowed the
production of pure DNA in sufficient quantities to allow its
chemical analysis. Another was the near perfection of gel
electrophoretic methods for high resolution separation of
oligonucleotides on the basis of their size. The key conceptual
development, however, was the introduction of methods of generating
size-nested sets of fragments cloned, purified DNA that contain, in
their collection of lengths, the information necessary to define
the sequence of the nucleotides comprising the parent DNA
molecules.
[0004] Two DNA sequencing methods are in widespread use. These are
the method of Sanger, F., Nicken, S. and Coulson, A. R. Proc. Natl.
Acad. Sci. U.S. Pat. No. 74, 5463 (1977) and the method of Maxam,
A. M. and Gilbert, W. Methods in Enzymology 65, 499-599 (1980).
[0005] The method developed by Sanger is referred to as the dideoxy
chain termination method. In the most commonly used variation of
this method, a DNA segment is cloned into a single-stranded DNA
phage such as M13. These phage DNAs can serve as templates for the
primed synthesis of the complimentary strand by the Klenow fragment
of DNA polymerase I. The primer is either a synthetic
oligonucleotide or a restriction fragment isolated from the
parental recombinant DNA that hybridizes specifically to a region
of the M13 vector near the 3" end of the cloned insert. In each of
four sequencing reactions, the primed synthesis is carried out in
the presence of enough of the dideoxy analog of one of the four
possible deoxynucleotides so that the growing chains are randomly
terminated by the incorporation of these "dead-end" nucleotides.
The relative concentration of dideoxy to deoxy forms is adjusted to
give a spread of termination events corresponding to all the
possible chain lengths that can be resolved by gel electrophoresis.
The products from each of the four primed synthesis reactions are
then separated on individuals tracks of polyacrylamide gels by the
electrophoresis. Radioactive tags incorporated in the growing
chains are used to develop an autoradiogram image of the pattern of
the DNA in each electrophoresis track. The sequence of the
deoxynucleotides in the cloned DNA is determined from an
examination of the pattern of bands in the four lanes.
[0006] The method developed by Maxam and Gilbert uses chemical
treatment of purified DNA to generate size-nested sets of DNA
fragments analogous to those produced by the Sanger method. Single
or double-stranded DNA, labeled with radioactive phosphate at
either the 3' or 5' end, can be sequenced by this procedure. In
four sets of reactions, cleavage is induced at one or two of the
four nucleotide bases by chemical treatment. Cleavage involves a
three-stage process: modification of the base, removal of the
modified base from its sugar, and strand scission at that sugar.
Reaction conditions are adjusted so that the majority of
end-labeled fragments generated are in the size range (typically 1
to 400 nucleotides) that can be resolved by gel electrophoresis.
The electrophoresis, auto-radiography, and pattern analysis are
carried out essentially as is done for the Sanger method. (Although
the chemical fragmentation necessarily generates two pieces of DNA
each time it occurs, only the piece containing the end label is
detected on the autoradiogram.)
[0007] Both of these DNA sequencing methods are in widespread use,
and each has several variations.
[0008] For each, the length of sequence that can be obtained from a
single set of reactions is limited primarily by the resolution of
the polyacrylamide gels used for electrophoresis. Typically, 200 to
400 bases can be read from a single set of gel tracks. Although
successful, both methods have serious drawbacks, problems
associated primarily with the electrophoresis procedure. One
problem is the requirement of the use of radio-label as a tag for
the location of the DNA bands in the gels. One has to contend with
the short half-life of phosphorus-32, and hence the instability of
the radio-labeling reagents, and with the problems of radioactive
disposal and handling. More importantly, the nature of
auto-radiography (the film image of a radioactive gel band is
broader than the band itself) and the comparison of band positions
between four different gel tracks (which may or may not behave
uniformly in terms of band mobilities) can limit the observed
resolution of bands and hence the length of sequence that can be
read from the gels. In addition, the track-to-track irregularities
make automated scanning of the auto-radiograms difficult--the human
eye can presently compensate for these irregularities much better
than computers can. This need for manual "reading" of the
auto-radiograms is time-consuming, tedious and error-prone.
Moreover, one cannot read the gel patterns while the
electrophoresis is actually being performed, so as to be able to
terminate the electrophoresis once resolution becomes insufficient
to separate adjoining bands, but must terminate the electrophoresis
at some standardized time and wait for the autoradiogram to be
developed before the sequence reading can begin.
[0009] The invention of the present patent application addresses
these and other problems associated with DNA sequencing procedures
and is believed to represent a significant advance in the art. The
preferred embodiment of the present invention represents a further
and distinct improvement.
SUMMARY OF THE INVENTION
[0010] Briefly, this invention comprises a novel process for the
electrophoetic analysis of DNA fragments produced in DNA sequencing
operations wherein chromophores or fluorophores are used to tag the
DNA fragments produced by the sequencing chemistry and permit the
detection and characterization of the fragments as they are
resolved by electrophoresis through a gel. The detection employs an
absorption or fluorescent photometer capable of monitoring the
tagged bands as they are moving through the gel.
[0011] This invention further comprises a novel process for the
electrophoretic analysis of DNA fragments produced in DNA
sequencing operations wherein a set of four chromophores are used
to tag the DNA fragments produced by the sequencing chemistry and
permit the detection and characterization of the fragments as they
are resolved by electrophoresis through a gel; the improvement
wherein the four different fragment sets are tagged with the
fluorophores fluorescein, Texas Red, tetramethyl rhodamine, and
7-nitro-benzofurazan.
[0012] This invention also includes a novel system for the
electrophoretic analysis of DNA fragments produced in DNA
sequencing operations comprising:
[0013] a source of chromophore or fluorescent tagged DNA
fragments.
[0014] a zone for containing an electrophoresis gel,
[0015] means for introducing said tagged DNA fragments to said
zone; and
[0016] photometric means for monitoring or detecting said tagged
DNA fragments as they move through and are separated by said
gel.
[0017] It is an object of this invention to provide a novel process
for the sequence analysis of DNA.
[0018] It is another object of our invention to provide a novel
system for the analysis of DNA fragments.
[0019] More particularly, it is an object of this invention to
provide an improved process for the sequence analysis of DNA.
[0020] These and other objects and advantages of this invention
will be apparent from the detailed description which follows.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Turning to the drawings:
[0022] FIG. 1 is an illustration of one means of end-labeling a DNA
fragment with a fluorescent tag. Pst. I and T4 DNA ligase are
enzymes commonly used in recombinant DNA research.
[0023] FIG. 2 is a block diagram of automated DNA sequencer, gel
electrophoretic system.
[0024] FIG. 3 is a comparison of the type of data produced by DNA
sequencing of the sequence shown in FIG. 1.
[0025] FIG. 4 is a block diagram of a preferred DNA sequencer
according to this invention.
[0026] FIG. 5 shows the emission spectra for the four fluorophores
used as tags in the preferred embodiment of this invention.
[0027] FIG. 6 is a schematic diagram of a possible optical
configuration in the detector unit. P, lamp source; L1, objective
lens; L2, collimating lens; F1, UV blocking filter; F2, heat
blocking filter; F3, band pass excitation filter; F4, long pass
emission filter; DM, dichroic mirror; C, polyacrylamide gel; PMT,
photomultiplier tube.
[0028] FIG. 7 is a schematic diagram of another possible optical
configuration in the detector unit. F1 to F4 are bandpass filters
centered at the emission maximum of the different dyes. P1 to P4
are photomultiplier tubes. The excitation light is of a wavelength
such that it is not transmitted through any of the filters F1 to
F4.
[0029] In the previous methods of DNA sequencing, including those
based on the Sanger dideoxy chain termination method, a single
radioactive label, phosphorus-32, is used to identify all bands on
the gels. This necessitates that the fragment sets produced in the
four synthesis reactions be run on separate gel tracks and leads to
the problems associated with comparing band mobilities in the
different tracks. This problem is overcome in the present invention
by the use of a set of four chromophores or fluorophores with
different absorption or fluorescent maxima, respectively. Each of
these tags is coupled chemically to the primer used to initiate the
synthesis of the fragment strands. In turn, each tagged primer is
then paired with one of the dideoxynucleotides and used in the
primed synthesis reaction with the Klenow fragment of DNA
polymerase.
[0030] The primers must have the following characteristics. 1) They
must have a free 3' hydroxyl group to allow chain extension by the
polymerase. 2) They must be complementary to a unique region 3' of
the cloned insert. 3) They must be sufficiently long to hybridize
to form a unique, stable duplex. 4) The chromophore or fluorophore
must not interfere with the hybridization or prevent 3' -end
extension by the polymerase.
[0031] Conditions 1, 2 and 3 above are satisfied by several
synthetic oligonucleotide primers which are in general use for
Sanger-type sequencing utilizing M13 vectors.
[0032] One such primer is the 15 mer 5' CCC AG TCA CGA CGT T 3'
where A, C, G and T represent the four different nucleoside
components of DNA; A, adenosine; C, cytosine; G, guanosine; T,
thymidine.
[0033] In the preferred embodiment of the present invention a set
of four fluorophores with different emission spectra, respectively,
are used. These different emission spectra are shown in FIG. 5.
Each of these tags is coupled chemically to the primer used to
initiate the synthesis of the fragment strands. In turn, each
tagged primer is then paired with one of the dideoxynucleotides and
used in the primed synthesis reaction with the Klenow fragment of
DNA polymerase.
[0034] The dyes used must have high extinction coefficients and/or
reasonably high quantum yields for fluorescence. They must have
well resolved adsorption maxima and/or emission masima.
Representative of such amino reactive dues are: fluorescein
isothiocyanage (FITC,
.lambda..sub.max.sup.=ex495,.lambda..sub.max.sup.em=520,
.epsilon..sub.4958.times.10.sup.4), tetramethyl rhodamine
isothiocyanate (TMRITC,
.lambda..sub.max.sup.ex=550,.lambda..sub.max.sup.Ex=578,.epsilon-
..sub.550 4.times.10.sup.4), and substituted rhodamine
isothiocyanate (XRITCA =580, .lambda..sub.max
Em=604,.epsilon.5808.times.10.sup.4) where .lambda. represents the
wavelength in nanometers, Ex is excitation, Em is emission, max is
maximum, and C is the molar extinction coefficient. These dyes have
been attached to the M13 primer and the conjugates electrophoresed
on a 20% polyacrylamide gel. The labeled-primers are visible by
both their-absorption and their fluorescence in the gel. All four
labeled primers have identical electrophoretic mobilities. The dye
conjugated primers retain their ability to specifically hybridize
to DNA, as demonstrated by their ability to replace the
underivitized oligonucleotide normally used in the sequencing
reactions.
[0035] The chemistry for the coupling of the chromophoric or
fluorophoric tags is described in assignee's copending patent
applications Ser. No. 565,010, filed Dec. 20, 1983, now abandoned,
and Ser. No. 709,579, filed Mar. 8, 1985, the disclosures of which
are expressly incorporated herein by reference. The strategy used
is to introduce an aliphatic amino group at the 5' terminus as the
last addition in the synthesis of the oligonucleotide primer. This
reactive amino group may then readily be coupled with a wide
variety of amino reactive fluorophores or chromophores. This
approach aids compatibility of the labeled primers with condition 4
above.
End Labeling of DNA for Use with Maxam/Gilbert Method
[0036] In the Maxam/Gilbert method of DNA sequencing, the end of
the piece of DNA whose sequence is to be determined must be
labeled. This is conventionally done enzymatically using
radioactive nucleosides. In order to use the Maxam/Gilbert method
in conjunction with the dye detection scheme described in this
invention, the DNA piece must be labeled with dyes. One manner in
which this maybe accomplished is shown in FIG. 1. Certain
restriction endonucleases generate what is known as a 3' overhang
as the product of DNA cleavage. These enzymes generate a "sticky
end, " a short stretch of single stranded DNA at the end of a piece
of double stranded DNA. This region will anneal with a
complementary stretch of DNA, which may be covalently joined to the
duplex DNA with the enzyme ligase. In this manner one of the
strands is covalently linked to a detectable moiety. This moiety
may be a dye, an amino group or a protected amino group (which
could be deprotected and reacted with dye subsequent to the
chemical reactions).
Sequencing reactions
[0037] The dideoxy sequencing reactions are performed in the
standard fashion Smith, A. J. H., Methods in Enzymology 65, 56-580
(1980), except that the scale may be increased if necessary to
provide an adequate signal intensity in each band for detection.
The reactions are done using a different color primer for each
different reaction. No radio-labeled nucleoside triphosphate need
be included in the sequencing reaction.
[0038] The Maxam/Gilbert sequencing reactions are performed in the
usual manner, Gil, S. F. Aldrichimica Acta 16(3), 59-61 (1983),
except that the end label is either one or four colored dyes, or a
free or protected amino group which may be reacted with dye
subsequently.
Detection
[0039] There are many different ways in which the tagged molecules
which have been separated by length using polyacrylamide gel
electrophoresis may be detected. Four illustrative modes are
described below. These are i) detection of the fluorescence excited
by light of different wave-lengths for the different dyes, ii)
detection of fluorescence excited by light of the same wavelength
for the different dyes, iii) elution of the molecules from the gel
and detection by chemiluminescence, and iv) detection by the
absorption of light by molecules. In modes i) and ii) the
fluorescence detector should fulfill the following requirements. a)
The excitation light beam should not have a height substantially
greater than the height of a band. This is normally in the range of
0.1 to 0.5 mm. The use of such a narrow excitation beam allows the
attainment of maximum resolution of bands. b) The excitation
wavelength can be varied to match the absorption maxima of each-of
the different dyes or can be a single narrow, high intensity light
band that excites all four fluorophores and does not overlap with
any of the fluorescence emission. c) The optical configuration
should minimize the flux of scattered and reflected excitation
light to the photodetector 14. The optical filters to block out
scattered and reflected excitation light are varied as the
excitation wavelength is varied. d) The photodetector 14 should
have a fairly low noise level and a good spectral response and
quantum efficiency throughout the range of the emission of the dyes
(500 to 600 nm for the dyes listed above). e) The optical system
for collection of the emitted fluorescence should have a high
numerical aperture. This maximizes the fluorescence signal.
Furthermore, the depth of field of the collection optics should
include the entire width of the column matrix.
[0040] Two illustrative fluorescence detection systems are
diagrammed in FIGS. 6 and 7. The system in FIG. 6 is compatible
with either single wavelength excitation or multi wavelength
excitation. For single wavelength excitation, the filter F4 is one
of four band pass filters centered at the peak emission wavelength
of each of the dyes. This filter is switched every few seconds to
allow continual monitoring of each of the four fluorophores. For
multi wavelength excitation, the optical elements F3 (excitation
filter), DM (dichroic mirror), and F4(barrier filter) are switched
together. In this manner both the excitation light and the observed
emission light are varied. The system in FIG. 7 is a good
arrangement for the case of single wave-length excitation. This
system has the advantage that no moving parts are required, and
fluorescence from all four of the dyes may be simultaneously and
continuously monitored. A third approach (iii above) to detection
is to elute the labeled molecules at the bottom of the gel, combine
them with an agent for excitation of chemiluminescence such as 1,2
dioxetane dione, Gill, S. K. Aldrichimica Acta 16(3), 59-61 (1983);
Mellbin, G. J. Liq. Chrom. 6(9), 1603-1616 (1983), and flow the
mixture directly into a detector which can measure the emitted
light at four separate wavelengths. The background signal in
chemiluminescence is much lower than in fluorescence, resulting in
higher signal to noise ratios and increased sensitivity. Finally,
the measurement may be made by measurements of light absorption (iv
above). In this case, a light beam of variable wavelength is passed
through the gel, and the decrease in the beam intensity due to
absorption of light at the different wavelengths corresponding to
the absorption maximum of the four dyes, it is possible to
determine which dye molecule is in the light path. As disadvantage
of this type of measurement is that absorption measurements are
inherently less sensitive than fluorescence measurements.
[0041] The above-described detection system is interfaced to a
computer 16. In each time interval examined, the computer 16
receives a signal proportional to the measured signal intensity at
that time for each of the four colored tags. This information tells
which nucleotide terminates the DNA fragment of the particular
length in the observation window at that time. The temporal
sequence of colored bands gives the DNA sequence. In FIG. 3 is
shown the type of data obtained by conventional methods, as well as
the type of data obtained by the improvements described in this
invention.
[0042] The following Examples are presented solely to illustrate
the invention. In the Examples, parts and percentages are by weight
unless otherwise indicated.
EXAMPLE I
Gel Electrophoresis
[0043] Aliquots of the sequencing reactions are combined and loaded
onto a 5% polyacrylamide column 10 shown in FIG. 2 from the upper
reservoir 12. The relative amounts of the four different reactions
in the mixture are empirically adjusted to give approximately the
same fluorescence or absorptive signal intensity from each of the
dye DNA conjugates. This permits compensation for differences in
dye extinction coefficients, dye fluorescence quantum yields,
detector sensitivities and so on. A high voltage is placed across
the column 10 so as to electrophorese the labeled DNA fragments
through the gel. The labeled DNA segments differing in length by a
single nucleotide are separated by electrophoresis in this gel
matrix. At or near the bottom of the gel column 10, the bands of
DNA are resolved from one another and pass through the detector 14
(more fully described above). The detector 14 detects the
fluorescent or chromophoric bands of DNA in the gel and determines
their color, and therefore to which nucleotide they correspond.
This information yields the DNA sequence.
EXAMPLE II
[0044] FIG. 4 shows a block diagram of a DNA sequenator for use
with one dye at a time. The beam (4880 A) from an argon ion laser
100 is passed into the polyacrylamide gel tube (sample) 102 by
means of a beamsteerer 104. Fluorescence exited by the beam is
collected using a low f-number lens 106, passed through an
appropriate set of optical filters 108 and 110 to eliminate
scattered excitation light and detected using a photomultiplier
tube (PMT) 112. The signal is readily detected on a strip chart
recorder. DNA sequencing reactions are carried out utilizing a
fluorescein labeled oligonucletide primer. The peaks on the chart
correspond to fragments to fluorescein labeled DNA of varying
lengths synthesized in the sequencing reactions and separated in
the gel tube by electrophoresis. Each peak contains on the order of
10.sup.-15 to 10.sup.-16 moles of fluorescein,.which is
approximately equal to the amount of DNA obtained per band in an
equivalent sequencing gel utilizing radioisotope detection. This
proves that the fluorescent tag is not removed or degraded from the
oligonucleotide primer in the sequencing reactions. It also
demonstrates that the detection sensitivity is quite adequate to
perform DNA sequence analysis by this means.
MATERIALS
[0045] Fluorescein-5-isothiocyanate (FITC) and Texas Red were
obtained from Molecular Probes, Inc. (Junction City, Oreg.).
tetramethyl rhodamine isothiocyanate (TMRITC) was obtained from
Research Organics, Inc. (Cleveland, Ohio).
4-fluoro-7-nitro-benzofurazan (NBD-fluoride) was obtained from
Sigma Chemical Co. (St. Louis, Mo.). Absorption spectra were
obtained on a H/P 8491 spectrophotometer. High performance liquid
chromatography was performed on a system composed of two Altex 110A
pumps, a dual chamber gradient mixer, Rheodyne injector, Kratos 757
UV detector, and an Axxiom 710 controller.
EXAMPLE III
Addition of 5'-Aminothymidine Phosphoramidites to
Oligonucleotides
[0046] The protected 5 '-aminothymidine phosphoramidites, 5'-(N-9
-fluorenylmethyloxycarbonyl) -5'-amino-5'-deoxy-3'-N,
N-diisopropylaminomethoxyphosphinyl thymidine, is coupled to the
5'-hydroxyl of an oligonucleotide using well established DNA
synthetic procedures. The solvents and reaction conditions used are
identical to those used in oligonucleotide synthesis.
EXAMPLE IV
Dye Conjugation
[0047] The basic procedure used for the attachment of fluorescent
dye molecules to the amino oligonucleotides is to combine the amino
oligonucleotide and the dye in aqueous solution buffered to pH 9,
to allow the reaction to stand at room temperature for several
hours, and then to purify the product in two stages. The first
purification step is to remove the bulk of the unreacted or
hydrolyzed dye by gel filtration. The second purification stage is
to separate the dye conjugate from unreacted oligonucleotide by
reverse phase high performance liquid chromatography. Slight
variations upon these conditions are employed for the different
dyes, and the specific procedures and conditions used for four
particular dyes are given below and in Table 1.
1TABLE 1 Reverse Phase HPLC Conditions for Dye-oligonucleotide
Purification Sample Retention time PLP-15.sup.a 18'
PLP-15-T-NH.sub.2.sup.b 18' FITC PLP-15.sup.c 27' NBD PLP-15 25'
TMRITC PLP-15 32' and 34'.sup.d Texas Red PLP-15 42'
[0048] Retention times shown are for HPLC gradients of 20% solvent
B/80% solvent A to 60% solvent B/40% solvent A in 40 min., where
solvent A is 0.1 M triethylammonium acetate pH 7.0 and solvent B is
50% acetonitrile, 50% 0.1 M triethylammonium acetate pH 7.0. The
column was an Axxiom ODS 5 micron C 18 column #555-102 available
from Cole Scientific, Calabasas, Calif. This gradient is not
optimized for purification of PLP-15 and PLP-15-T-NH.sub.2, but the
retention times are included for comparison with the dye primer
conjugates.
[0049] .sup.aPLP-15 is an oligonucleotide primer for DNA sequence
analysis in the M13 vectors. Its sequence is 5' CCC AGT CAC GAC FTT
3'.
[0050] .sup.bPLP-15-T-NH.sub.2 is the oligonucleotide PLP-15 to
which a 5'-amino-5'-deoxythymidine base has been added to==at the
5' terminus.
[0051] .sup.cthe nomenclature Dye PLP-15 signifies the conjugate of
PLP-15-T-NH.sub.2 and the dye molecule.
[0052] .sup.dTwo fluorescent oligonucleotide products were obtained
with TMRITC. Both were equally effective in sequencing. This is
presumed to be due to the two isomers of TMRITC which are present
in the commercially available material.
[0053] The following procedure is for use with fluorescein
isothiocyanate or 4-fluoro-7-nitro-benzofurazan. Amino
oligonucleotide (0.1 ml of .about.1 mg/ml oligonucleotide in water)
is combined with 1M sodium carbonate/bicarbonate buffer pH 9 (50
.mu.l) 10 mg/ml dye in dimethylformamide (20 .mu.l) and H.sub.2O
(80 .mu.l). This mixture is kept in the dark at room temperature
for several hours. The mixture is applied to a 10 ml column of
Sephadex G-25 (medium) and the colored band of material eluting in
the excluded volume is collected. The column is equilibrated and
run in water. In control reactions with underivatized
oligonucleotides, very little if any dye is associated with the
oligonucleotide eluting in the void volume. The colored material is
further purified by reverse phase high performance liquid
chromatography on an Axxiom C.sub.18 column (#555-102, Cole
Scientific, Calabasas, Calif.) in a linear gradient of
acetonitrile:0.1M triethylammonium acetate, pH 7.0. It is
convenient for this separation to run the column eluant through
both a UV detector (for detecting the DNA absorbance) and a
fluorescence detector (for detecting the dye moiety). The desired
product is a peak on the chromatogram which is both strongly UV
absorbing and strongly fluorescent. The dye oligonucleotide
conjugates elute at higher acetonitrile concentrations than the
oligonucleotides alone, as shown in Table 1. The oligonucleotide is
obtained from the high performance liquid chromatography in
solution in a mixture of acetonitrile and 0.1M triethylammonium
acetate buffer. This is removed by lyophilization and the resulting
material is redissolved by vortexing in 10 mM sodium hydroxzide
(for a minimum amount of time) followed by neutralization with a
five fold molar excess (to sodium hydroxide) of Tris buffer, pH
7.5.
[0054] The conjugation with Texas Red is identical to that
described for fluorescein isothiocyanate and
4-fluoro-7-nitro-benzofurazan, except that:
[0055] a) prior to separation on Sephadex G-25 the reaction is made
1M in ammonium acetate and kept at room temperature for 30 minutes,
and
[0056] b) the Sephadex G-25 column is run in 0.1 M ammonium
acetate. This largely eliminates nonspecific binding of the dye
molecule to the oligonucleotide.
[0057] The conjugation with tetramethyl rhodamine isothiocyanate is
identical to that for Texas Red except that the reaction-is carried
out in 10 mM sodium carbonate/bicarbonate buffer, pH 9.0, and 50%
dioxane. This increases solubility of the tetramethyl rhodamine and
a much higher yield of dye oligonucleotide conjugate is
obtained.
[0058] In some cases, particularly with the rhodamine-like dyes, a
substantial amount of nonspecific binding of dye was observed, as
manifested by an inappropriately large dye absorption present in
the material eluted from the gel filtration column. In these cases
the material was concentrated and reapplied to a second gel
filtration column prior to high performance liquid chromatography
purification. This generally removed the majority of the
noncovalently associated dye.
EXAMPLE V
Properties of Dye-oligonucleotide Conjugates
[0059] The development of chemistry for the synthesis of dye
oligonucleotide conjugates allows their use as primers in DNA
sequence analysis. Various fluorescent dye primers have been tested
by substituting them for the normal primer in DNA sequence analysis
by the enzymatic method. An autoradiogram of a DNA sequencing gel
in which these dye-conjugated primers were utilized in T reactions
in place of the normal oligonucleotide primer was prepared. This
autoradiogram was obtained by conventional methods employing
.alpha.-.sup.32P-dCTP as a radio-label. The autoradiogram showed
that the underivitized primer, amino-derivitized primer, and dye
conjugated primers all give the same pattern of bands
(corresponding to the DNA sequence), indicating that the
derivitized primers retain their ability to hybridize specifically
to the complementary strand. Secondly, the bands generated using
the different primers differ in their mobilities, showing that it
is indeed the dye-primers which are responsible for the observed
pattern, and not a contaminant of unreacted or underivitized
oligonucleotide. Thirdly, the intensity of the bands obtained with
the different primers is comparable, indicating that the strength
of hybridization is not significantly perturbed by the presence of
the dye molecules.
[0060] The separations are again carried out in an acrylamide gel
column. The beam from an argon ion laser is passed into the
polyacrylamide gel tube (sample) by means of a beam-steerer.
Fluorescence exited by the beam is collected using a low f-number
lens, passed through an appropriate set of optical filters to
eliminate scattered excitation light and detected using a
photomultiplier tube (PMT). The signal is monitored on a strip
chart recorder. DNA sequencing reactions have been carried out
utilizing each of the four different dye coupled oligonucleotide
primers. In each case a series of peaks are observed on the chart
paper. The peaks correspond to fragments of dye labeled DNA of
varying lengths synthesized in the sequencing reactions and
separated in the gel tube by electrophoresis. Each peak contains of
the order of 10.sup.-14 to 10.sup.-16 moles of dye, which is
approximately equal to the amount of DNA obtained per band in an
equivalent sequencing gel utilizing radioisotope detection. This
proves that the fluorescent tag is not removed or degraded from the
oligonucleotide primer in the sequencing reactions. It also
demonstrates that the detection sensitivity is quite adequate to
perform DNA sequence analysis by this means, and that adequate
resolution of the DNA fragments is obtained in a tube gel
system.
[0061] Having fully described the invention it is intended that it
be limited only by the lawful scope of the appended claims.
* * * * *