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Claims  |
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What is claimed is:
1. An apparatus for performing DNA sequencing, comprising:
a) a body comprising a substrate and a cover plate bonded thereto wherein
said coverplate is transparent;
b) a channel pattern formed within said body and including at least one gel
channel having a first and second longitudinal ends and being at least
partially filled with gel;
c) a reaction chamber in communication with said gel and having attached
thereto photocleavable linkers wherein said photocleavable linkers have
bound thereto oligonucleotide probes/primers;
d) means for communicating a solution containing at least one target DNA to
said reaction chamber for hybridization with said DNA synthesis primers;
and
e) means for establishing an electric field between said first and second
longitudinal ends of said at least one gel channel, of sufficient strength
to impart electrophoretic separation of chain-terminated strands of DNA
through said gel.
2. The apparatus of claim 1, wherein said body comprises either silicon,
glass, quartz, plastic, or ceramics.
3. The apparatus according to claim 2, wherein the substrate is made of
optically transparent material.
4. The apparatus according to claim 1, wherein said channel pattern
includes a plurality of gel channels, and said communicating means
comprises at least one manifold connecting channel interconnecting said
plurality of gel channels at one common end.
5. The apparatus according to claim 1, wherein said means for establishing
an electric field comprises first and second electrodes disposed
respectively at said first and second opposite ends of said at least one
gel channel, and a d.c. power source coupled to said first and second
electrodes.
6. The apparatus according to claim 1 wherein said cover plate has access
ports for the introduction of substances into said reaction chamber.
7. A method of making a nucleic acid sedquencing microchip apparatus
comprising the steps of:
a) forming a channel pattern in a planar surface of a substrate, said
channel pattern including at least one gel channel;
b) fixedly attaching a cover plate on said planar surface of said
substrate;
c) partially filling said at least one gel channel with a gel, and thereby
forming a reaction chamber in the untilled portion wherein said reaction
chamber has attached thereto photocleavable linkers wherein said
photocleavable linkers have bound thereto oligonucleotide probes/primers;
and
d) placing first and second electrodes respectively at opposite ends of
said at least one gel channel.
8. The method according to claim 7, wherein said step of partially filling
said at least one gel channel includes filling said at least one gel
channel with an acrylamide buffer solution, covering said reaction chamber
portion of said gel channel with a photo-blocker, photopolymerizing said
acrylamide solution within said parallel channels with a light source,
whereby acrylamide in said reaction chamber is not polymerized, and then
flushing out the remaining acrylamide.
9. The method according to claim 8, wherein said filling step includes
electro-osmotically pumping said acrylamide buffer solution into said at
least one gel channel.
10. The method according to claim 7, wherein said step of forming a channel
pattern includes etching the channel pattern into, said planar surface of
said substrate using photolithographic techniques.
11. The method according to claim 7, wherein said step of forming a channel
pattern includes micro-machining said channel pattern.
12. The method according to claim 7, wherein said step of forming a channel
pattern includes forming a plurality of parallel gel channels, and forming
at least one manifold connecting channel that interconnects said plurality
of parallel gel channels.
13. An apparatus for simultaneously sequencing a plurality of polynucleic
acids comprising:
a) a solid support substrate;
b) a plurality of sequencing channels, each said channel having a reaction
well and a separating zone;
c) means for delivering substances to each said reaction well;
d) means for specifically selecting and binding a predetermined target
sequence within said reaction well of each said channel;
e) means for individually controlling sequencing reactions in each said
reaction well;
f) photocleavable linkers bound to said reaction wells and wherein said
photocleavable linkers have bound thereto oligonucleotide probes/primers;
g) means for applying an electrophoretic voltage across separating zone;
h) means for monitoring said electrophoretic separation to determine a
sequence of a polynucleic acid from the target sequence in each said
reaction well; and
i) a transparent cover plate fixedly attached to said substrate.
14. The apparatus according to claim 13, wherein said solid support
substrate is a glass microchip.
15. The apparatus according to claim 13, wherein said plurality of
sequencing channels are formed by etching.
16. The apparatus of according to claim 13, wherein said means for
delivering substances to each of said reaction wells comprises at least
one manifold connecting channel that intersects each of said reaction
wells.
17. The apparatus according to claim 16, wherein said means for delivering
substances to each said reaction well further comprises an additional
manifold connecting channel that intersects each of said reaction wells
and being disposed between said reaction well and said separation zone.
18. The apparatus according to claim 16, wherein said means for delivering
substances to each said reaction well further comprises electro-osmotic
means for pumping reaction compounds through said manifold connecting
channel to said reaction well of each said sequencing channel.
19. The apparatus according to claim 13, wherein said means for
specifically selecting and binding a predetermined target sequence within
said reaction well of each said sequencing channel comprises a linker
compound covalently bonded to a primer compound wherein said primer
compound is capable of priming a synthetic reaction.
20. The apparatus according to claim 13, wherein said means for
specifically selecting and binding a predetermined target sequence within
said reaction well of each said sequencing channel comprises a
probe/primer compound complementarily hydrogen bonded to a linker
compound.
21. The apparatus according to claim 13, wherein said means for applying
electrophoretic voltage comprises first and second electrodes disposed at
opposite ends of said plurality of sequencing channels.
22. The apparatus according to claim 13, wherein said plurality of
sequencing channels number between 50 and 10,000.
23. The apparatus according to claim 13, wherein each sequencing channel is
between 10 and 100 .mu.m in width.
24. The apparatus according to claim 13, wherein said means for monitoring
said electrophoretic separation comprises plural fluorescent labels, each
of said fluorescent labels indicating one nucleotide base, where said
nucleotide base is an element of the sequence of the polynucleic acid of
said reaction well.
25. The apparatus according to claim 13 wherein said cover plate has access
ports for the introduction of substances into said reaction wells. |
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Claims |
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Description |
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FIELD OF THE INVENTION
The present invention relates generally to sequencing polynucleotides and
more specifically to the simultaneous parallel sequence determination of
the sequences of a large plurality of polynucleotides. Arrays of
oligonucleotide probes are covalently bonded to multiple parallel lanes
etched into a glass microchip device. Hybridization, synthesis and
subsequent electrophoresis all occur within the one device to determine
the large plurality of sequences.
Each array comprises multiple copies of a species of oligonucleotide
primers. The primers act similar to an oligonucleotide probe, used in
Southern or Northern blotting, to select the target polynucleotide for
sequencing and the same oligonucleotide species strand also serves as a
primer for the synthesis of a nucleotide strand. Each newly synthesized
strand is complementary to the target polynucleotide by hydrogen bonding
between opposite strand basepairs. Any template based sequencing reaction
can be used, including Maxam-Gilbert or Sanger reactions.
BACKGROUND OF THE INVENTION
Since the cracking of the genetic code in the middle of the twentieth
century, determining the base sequences of DNA and RNA has been a tool for
elucidating the primary structure of peptides and proteins. Sequence
information is also useful for determining gross polynucleotide structure
and control of gene expression. The base sequences of non-coding
polynucleotide regions are also useful for studying mutation events,
phylogenetic linkages, polynucleotide structural characteristics, cell
cycle control, cancer and transcription and translation mechanisms.
Two sequencing methods are commonly used: the Maxam-Gilbert, or chemical
degradation method, and the Sanger, or dideoxy terminator or enzymatic
method. Either method delivers a family of DNA strands. Each strand
species is incrementally longer by one base than the next smaller species.
By tagging the strands to indicate which nucleotide is additional to the
next smaller strand species, the sequence of bases of the polynucleotide
can be determined. Gel electrophoresis is commonly used to resolve the
different lengths for analysis and determination of sequence.
During the sequencing reaction each polynucleotide strand can be tagged by
labelling the primers, by labelling the terminal base itself, or by
labelling a plurality of one base incorporated into each strand.
Two common labelling methods are the use of radioisotopes and fluorescent
tags. Using a different fluorescent tag for each terminal base allows
sequencing analysis to be accomplished in a single electrophoresis lane.
Other methods require multiple parallel lanes for sequencing one
polynucleotide fragment.
For example, the original Sanger method required four (4) parallel reaction
vessels. Each vessel was identical except for the terminating dideoxy base
included in the reaction mixture. Thus when products from the four vessels
were electrophoresed in four parallel lanes, each lane revealed only the
DNA strands terminating in the respective dideoxy base. By comparing the
four lanes containing a DNA ladder of lengths of DNA differing by only one
base and knowing the terminating base of each lane, the sequence could be
determined.
Another method being developed uses non-radioactive isotopic labels and
mass spectrometry to determine the polynucleotide sequence.
At present the most automated systems use either Sanger or Maxam-Gilbert
sequencing chemistry, and tag the resultant DNA species with fluorescent
probes. On line detection is accomplished as each band is electrophoresed
past a detection window. Commercial embodiments of this technology,
however, are limited to thirty-six or fewer simultaneous sequence
determinations per electrophoresis plate.
An automated electrophoresis apparatus is described in U.S. Pat. No.
5,279,721 to Schmid. Molecules are electrophoretically separated, based on
molecular weight, by a horizontal electric field. An impermeable sheet is
then removed allowing a vertical electrical field to effect transfer of
the separated substances to a blot membrane.
While automating some aspects of electrophoresis and electroblotting, the
apparatus described in U.S. Pat. No. 5,279,721 does not sequence a
polynucleotide or provide means for the required multiple serial
reactions. Rather, it addresses Southern blotting procedures wherein
specific nucleotide sequences are detected by complementary binding with a
probe nucleotide strand.
Another method, described in U.S. Pat. No. 5,302,509 to Cheeseman, uses a
solid support to anchor a DNA template to the apparatus and determines
each complementary base species as it is added during the synthesis
process. This method does not describe gel electrophoresis for separation.
Solid phase supports are also described in WO 93/20232. Here two or more
regions of target DNA could be sequenced by annealing them to opposite
selective sequencing primers. A modified Sanger reaction followed. In a
preferred embodiment formamide was used to chemically melt the DNA from
the Dynabead supports before electrophoresis into the separating gel. This
method lends itself to PCR amplification of very small quantities of DNA
prior to the sequencing reactions.
These and other sequencing schemes are advancing due to the impetus of the
human genome project. The goal of the genome project, to sequence the
entire human genome (and selected genes of other species) has been likened
to the 1960's era space program to put a man on the moon by the end of the
decade. Many researchers are therefore proposing methods to rapidly and
inexpensively sequence massive lengths of genetic materials. Reducing the
costs and errors inherent in human manipulations is a common thread of
these proposals.
SUMMARY OF THE INVENTION
The present invention provides a novel microchip based apparatus and method
for sequencing massively multiple polynucleotide strands with a minimal
requirement for human intervention. Techniques borrowed from the
microelectronics industry are particularly suitable to these ends.
Micromachining and photolithographic procedures are capable of producing
multiple parallel microscopic scale components on a single chip substrate.
Materials can be mass produced and reproducibility is exceptional. The
microscopic sizes minimize material requirements.
Human manipulations can be minimized by designing and building a dedicated
apparatus capable of performing a series of functions. For example, DNA
sequencing requires: 1) selection and purification of the target DNA
strand, 2) labeling the strand in a manner to permit sequencing, 3)
producing a family of strands beginning with a specified base from the
target DNA strand and terminating at every incremental base along the
sequencing region of the strand to be sequenced, 4) separating a mixture
of strands differing in length by one base, and 5) identifying the last
incremental base.
The present invention proposes a novel microchip based apparatus and method
for accomplishing these procedures on the single microchip. Sequencing
massively multiple polynucleotide strands economically and with a minimal
requirement for human intervention is thus feasible.
An object of the present invention is to provide a massively parallel
automated DNA sequencing method and apparatus in which multiple serial
reactions are automatically performed individually within one reaction
well for each of the plural polynucleotide strands to be sequenced in the
plural parallel sample wells. These serial reactions are performed in a
simultaneous run within each of the multiple parallel lanes of the device.
"Parallel" as used herein means wells identical in function.
"Simultaneous" means within one preprogrammed run. The multiple reactions
automatically performed within the same apparatus minimize sample
manipulation and labor.
A further object of this invention is to provide an apparatus and method
wherein a plurality of samples can be simultaneously processed to
determine a polynucleotide sequence for each sample.
Another object of the invention is to provide selection means within each
well to uniquely select the target polynucleotide to be sequenced.
Yet another object of the present invention is to provide a means for
segregating sequencing reactions in a small undivided volume, thereby
conserving reagents and enzymes.
Another object of the present invention is to provide a miniaturized
electrophoresis system for separation of nucleotides or other molecules
based on their electrophoretic mobilities. The miniaturization allows
sequence determination to be accomplished using very small amounts of
sample.
Still another object of the invention is to provide multiple reaction
wells, the reaction wells being reaction chambers, on a microchip, each
reaction well containing an individualized array to be used for
determining the nucleotide sequence uniquely specified by the substrates
provided, the reaction conditions and the sequence of reactions in that
well.
Some objects of Applicant's invention are met by an apparatus for
performing DNA sequencing comprising a body, a channel pattern formed
within the body and including at least one gel channel. The gel channel
has first and second longitudinal ends and are at least partially filled
with gel. The apparatus further comprises a reaction chamber in
communication with the gel and having a surface for attachment of DNA
synthesis primers. The apparatus further comprises means for communicating
a solution containing at least one target DNA to the reaction chamber for
hybridization with the DNA synthesis primers, and means for establishing
an electric field between the first and second longitudinal ends of the at
least one gel channel, of sufficient strength to impart electrophoretic
separation of chain-terminated strands of DNA through the gel.
Other objects of Applicant's invention are met by a method of making a
microchip used in making a DNA sequencing analysis comprising the steps of
a) forming a channel pattern in a planar surface of a substrate wherein
the channel pattern includes at least one gel channel; b) fixedly
attaching a planar surface of a cover plate on the planar surface of the
substrate; c) partially filling the at least one gel channel with a gel
and thereby forming a reaction chamber in the unfilled portion; and d)
placing first and second electrodes respectively at opposite ends of the
at least one gel channel.
Still, other objects are met by an apparatus for simultaneously sequencing
a plurality of polynucleic acids comprising a solid support substrate, a
plurality of sequencing channels wherein each channel has a reaction well
and a separating zone. The apparatus further comprises means for
delivering and flushing substances to each reaction well; means for
specifically selecting and binding a predetermined target sequence within
the reaction well of each channel; means for individually controlling
sequencing reactions in each reaction well; means for detaching
polynucleotide strands from the solid support substrate; means for
applying an electrophoretic voltage across the separating zone; and means
for monitoring the electrophoretic separation to determine a sequence of a
polynucleic acid from the target sequence in each reaction well.
In accordance with yet other objects of Applicant's invention, a method for
sequencing polynucleic acids comprises the steps of a) placing a DNA probe
capable of acting as a DNA synthesis primer in a reaction chamber
juxtaposed at least one gel electrophoresis lane; b) introducing into the
reaction chamber a target DNA containing a sequence complementary to the
DNA probe, and thereby allowing the DNA probe and the target DNA to
hybridize; c) introducing into the reaction chamber reagents, labeling
means and enzymes for dideoxy sequencing, and allowing sequencing
reactions to form labelled terminated chains; d) separating the labelled
terminated chains from the target DNA; and e) applying a voltage across at
least one gel electrophoresis lane to electrophoretically separate the
labelled terminated chains.
Other objects are met by a method for sequencing polynucleic acids
comprising the steps of a) placing a DNA probe capable of selecting a
target DNA sequence in a reaction chamber juxtaposed at least one gel
electrophoresis lane; b) introducing into the reaction chamber a target
DNA containing a sequence complementary to the DNA probe, and thereby
allowing the DNA probe and the target DNA to hybridize; c) introducing
into the reaction chamber reagents, and labeling means for chemical
degradation sequencing, and allowing sequencing reactions to form
shortened sequencing chains; d) separating the shortened sequencing chains
from the probe DNA; and e) applying a voltage across the at least one gel
electrophoresis lane to electrophoretically separate the shortened
sequencing chains.
In accordance with still yet other objects of Applicant's invention, a
method of sequencing a target DNA sequence comprises the steps of a)
placing a plurality of probe/primers respectively in corresponding
reaction chambers, each probe/primer having a unique and defined sequence
which allows the probe/primer to hybridize to a specific sequence, at
different locations, in the target DNA and to act as a primer for DNA
sequencing reactions; b) forming a gel channel respectively in
communication with corresponding ones of the reaction chambers; c) adding
a solution containing the target DNA sequence to the reaction chambers; d)
adding reagents and labeled enzymes to permit dideoxy reactions which form
chain terminated labeled strands initiated at different locations of the
target DNA; e) separating labeled strands; and f) analyzing the separated
strands for DNA sequence.
The invention accomplishes the aforementioned operations on a single device
with a minimum of human intervention and handling. The microchip is
preferably transparent, thereby allowing photochemical reactions to be
controlled individually within each of the large plurality of lanes using
automated equipment and a preprogrammed protocol and also allowing
detection of fluorescently labelled strands.
Other objects, advantages and salient features of the invention will become
apparent from the following detailed description, which taken in
conjunction with the annexed drawings, discloses preferred embodiments of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a top schematic view of a microchip according to a preferred
embodiment of the present invention;
FIG. 2 is an end view of the microchip of FIG. 1;
FIG. 3 is an enlarged view of the area delineated by the broken line
rectangle of FIG. 1;
FIGS. 4(a)-(d) sequentially illustrate the sequencing methodology of the
present invention;
FIGS. 5(a) and 5(b) are enlarged views of a labeled, dideoxy terminated
strand 40L before and after denaturing, respectively.
FIGS. 6(a)-(f) sequentially illustrate a sequencing method of an
alternative embodiment of the present invention, using photocleaving;
FIG. 7 is a view similar to FIG. 3, showing an alternative embodiment,
where a single oligonucleotide sequence acts as a linker/probe/primer and
has a photocleavable linker group; and
FIG. 8 is a schematic view showing sequencing of a single target DNA strand
at different locations using different probe/primers.
DETAILED DESCRIPTION OF THE INVENTION
Referring to FIGS. 1 and 2, a microchip 10 for performing large-scale
microsequencing of DNA includes a substrate 12 and a cover plate 14 bonded
thereto. A channel pattern, etched into an upper surface of the substrate,
includes a plurality of parallel channels 16 and 18 interconnected for
fluid communication at a common end through at least one manifold
connecting channel 20. The number of parallel channels can range from one
to 1,000 or more.
The substrate 12 is preferably made of biocompatible material that is
transparent to light, including glass and quartz. Silicon, plastics and
ceramics may also be used. In one particular embodiment, the substrate 12
is a 3 cm long by 1 cm wide by 0.25 cm thick microscope slide. The cover
plate 14 is also preferably transparent and may, for example, be a
microscope slide or cover slip of similar dimensions to the substrate 12.
The channel pattern is formed in the glass substrate 12 using standard
photolithographic procedures followed by chemical wet etching. Standard
photolithographic procedures include transferring the pattern to the
substrate using a positive photoresist (Shipley 1811) and an e-beam
written chrome mask (Institute of Advanced Manufacturing Sciences, Inc.).
Wet etching includes using a HF/NH.sub.4 F solution. A similar channel
forming technique can be found in a patent application by Ramsey to a
microminiature capillary zone electrophoresis apparatus, Ser. No.
08/283,769, incorporated herein by reference. Micromachining techniques
may alternatively be employed to form the channel pattern.
The parallel channels 16 and 18 are approximately 50 .mu.m in width and
approximately 10 to 20 .mu.m in depth. The length can be longer than the
glass substrate and up to a few cm by forming the channels in a serpentine
shape. A thousand or more parallel channels can be formed on the microchip
as described herein. The manifold connecting channel 20 is perpendicular
to the parallel channels and is 50 to 100 .mu.m wide and approximately 10
to 20 .mu.m in depth.
After forming the channel pattern on the substrate 12, the cover plate 14
is bonded to the top surface of the substrate 12 using a direct bonding
technique whereby the cover plate 14 and the substrate 10 are first
hydrolyzed in dilute NH.sub.4 OH/H.sub.2 O.sub.2 solution and then joined.
The assembly is then annealed at about 500.degree. C. in order to insure
proper adhesion of the cover plate 14 to the substrate 12. The cover plate
14 thus encloses the channels etched (machined, or otherwise formed) in
the upper surface of the substrate 12.
In the preferred embodiment, an acrylamide buffer solution is
electro-osmotically pumped through the manifold connecting channel 20 into
the parallel channels 16 and 18. A volume of separating gel 22 and 24 is
formed within each parallel channel 16 and 18, respectively, by
photopolymerizing the acrylamide solution within the parallel channels.
Acrylamide in the manifold connecting channel 20 and an approximately 50
.mu.m length of each parallel channel at the intersection with the
manifold connecting channel 20 is not photo-irradiated so that the
acrylamide contained therein is not polymerized. The non-polymerized
acrylamide can be flushed from the system with an appropriate wash buffer.
Flushing can be accomplished by electro-osmotic pumping, by
electrophoretic pumping or by a pressure gradient. Typical electric field
strengths range from 200 to 700 V/cm but may range from 50 to 1500 v/cm.
Approximately 50 .mu.m wide by 50 .mu.m long by 20 .mu.m deep reaction
wells 26 and 28 are thus formed at one end portion of each parallel
channel 16 and 18, respectively. Most of the remaining length of each
parallel channel 16 and 18 provides a separating zone which is used for
electrophoretic separation of the products formed in the reaction wells,
reaction chambers, 26 and 28.
First and second electrodes 30 and 32 are disposed, respectively, in the
manifold connecting channel and at opposite ends of the parallel channels
16 and 18. The second electrode 32 is shown in direct contact with the gel
22 and 24 but may be separated from it by an electrically conducting
solution. An electric potential established at the two electrodes, and the
electric field established therebetween, provides the motive force for
electrophoretic manipulations, to be described more fully below.
The manifold connecting channel 20 is used for delivering polynucleotides,
reagents, wash solutions, buffers and the like to the reaction wells 26
and 28. Additional manifold connecting channels can be used also for
delivering additional reagents, wash solutions, buffers and the like to
the reaction wells. In the preferred embodiment, the ends of the manifold
connecting channel are connected to fluid reservoirs and the various
solutions, reagents and the like are electro-osmotically or
electrophoretically transported through the channel to the reaction wells
by applying electric potentials to reservoirs at opposite ends of the
channel. Alternatively, a pressure gradient may be used to transport
solutions through the channel. Reagents, etc. are added at one end of the
channel and wastes are collected at the other end. In an alternative
embodiment, a second manifold connecting channel, parallel to the first
manifold connecting channel 20 from FIGS. 1 and 2 may be disposed between
the reaction wells and the separating zones of each of the sequencing
channels. Reagents, wash solutions, etc. may then be transported from one
of the manifold channels to the other manifold channel through the
reaction wells by applying an electric potential difference or a pressure
gradient between the two channels. In a further alternative embodiment,
the cover plate 14 may be provided with access ports allowing the use of
micropipetting procedures, preferably robotic, to introduce substances
into the reaction wells. Each reaction well 26 and 28 acts as a
hybridization/synthesis chamber, a reaction chamber and contains
probe/primer oligonucleotides of defined sequence, and immobilized as
shown in FIG. 3. As seen therein, a linker 34, preferably an alkyl chain
10 to 20 carbons in length, is shown covalently attaching the 3' end of a
oligonucleotide strand 36 to a surface 38 of the substrate 12 (or cover
plate 14). Surface 38 is a longitudinal sidewall of the parallel channel
16. Several such alkyl linkers 34 are provided in each reaction well, as
shown in FIG. 1.
The linker oligonucleotides 36 in each specific reaction well have a unique
sequence which allows them to hybridize with the 5' end of a specific
probe/primer oligonucleotide 40 via hydrogen-bonding of complementary base
pairs according to Watson-Crick base-pair rules. The 3' overhanging end of
the probe/primer 40 also has a unique sequence which allows it to
hybridize to a specific sequence in the target DNA and to act as a primer
for DNA sequencing reactions.
The alkyl linkers may be attached through a Si--C direct bond or through an
ester, Si--O--C, linkage Maskos and Southern, Nucleic Acids Research, 20:
1679-1684, 1992). The alkyl linker may contain backbone atoms other than
carbon to provide additional reaction or cleavage sites. An example using
a 2-nitrobenzyl derivative for photocleaving means is described by Pillai
in Organic Photochemistry, Vol. 9, Albert Padwa, Ed., Marcel Dekker, Inc.
pp. 225-323, 1987, incorporated herein by reference.
An alternate method for attaching oligonucleotides to glass is described by
Graham et al. See Biosensors & Electronics, 7: 487-493, 1992.
As many as 10.sup.6 linker oligonucleotides per .parallel.m.sup.2 may be
attached, though steric considerations may warrant a lower density. These
oligonucleotides are preferably synthesized in situ by photochemical
methods, examples of which are described in Organic Photochemistry.
A simple binary method allows multiple simultaneous, spatially localized,
parallel syntheses of 2.sup.n compounds in n steps is described by Fodor,
et al., Science 251: 767-773, 1991. For example approximately 65,000
different oligonucleotides, the total number of 8-mers using four
nucleotide bases, could be synthesized by these techniques is 32 chemical
steps. On a smaller scale the approximately 1000 unique 5-mers could be
synthesized in 20 steps.
The photochemistry involves protection of the 5' hydroxyl group of
nucleosides or nucleotides with a protecting group to prevent undesirable
chemical reactions. The protecting group preferably is a selectively
removable chemical compound. Most preferably it is photolabile. The group
can be any moiety which undergoes photolysis to regenerate the 5' alcohol.
Groups suitable for blocking may be found in: Pillai, Photolytical
Deprotection and Activation of Functional Groups, Organic Photochemistry,
Albert Padwa Ed. Vol. 19 Chapter 3, pp. 225-323, 1987; Pillai,
Photoremovable Photoprotecting Groups in Organic Synthesis, Synthesis,
1980 p. 1-26; Organic Synthesis a Practical Approach, M. J. Gait Ed. IRl
Press, Oxford, Washington, D.C., 1984; incorporated herein by reference.
Such a synthesis protocol using nominal 50 .mu.m lanes allows packing
approximately 100 lanes each with a different oligonucleotide for each
linear cm of glass substrate. A plurality of sequencing channels can
number from between 50 and 10,000 and each sequencing channel can range
from 10 to 100 .mu.m in width.
As seen in FIG. 3, the probe/primer oligonucleotide 40 is shown attached by
its 5' end portion, to the 5' end portion of the linker oligonucleotide 36
by complementary hydrogen bonding. Preferably, the linker oligonucleotide
36 is 10 to 15 nucleotides in length and is complementary to approximately
the same number of nucleotides at the 3' terminal portion of the
probe/primer oligonucleotide 40.
The 3' end portion of the probe/primer 40 includes a unique sequence of 8
to 20 nucleotides complementary to the 3' terminal portion of the target
sequence. The unique sequence of the probe/primer 3' terminal portion
uniquely selects the target polynucleotide for processing in each reaction
well. An oligonucleotide eight bases in length has .about.65,000 different
possibilities using one of four possible bases at each position.
Similarly, an oligonucleotide fifteen bases in length has over 1 billion
different sequences possible.
Sequencing Methodology
FIGS. 4(a)-(d) illustrates sequentially the steps undertaken to effect a
DNA sequencing operation. As seen in FIG. 4(a), a solution containing
different target DNA sequences "A" and "B" is added to reaction wells 26
and 28 through the manifold connecting channel 20. The direction of flow
is indicated by the directional arrows.
In FIG. 4(b), A and B hybridize to specific probe/primers in reaction wells
26 and 28, respectively. In particular, the target sequence specific to
the each of the reaction wells 26 and 28 are selected from a mixture of
target sequences by complementary hydrogen bonding of the 3' end portion
of the probe/primer 40 to the 3' end portion of the target sequence A or
B. The 3' end of the probe/primer serves as a primer site for
polynucleotide synthesis using the target sequence as a template.
Once each probe/primer is bound to its specific target sequence,
non-hybridized DNA is eluted by flushing the reaction wells. The eluted
DNA molecules include extra copies of the target sequence, the target
sequences specified by the probe/primers in the other wells, and sequences
not targeted.
In the next step, FIG. 4(c), reagents and enzyme for dideoxy (Sanger)
sequencing are added and sequencing reactions are allowed to occur. The
reactions are manifest by elongation (shown as heavy line segments) of the
probe/primers 40, which terminate via dideoxy sequencing methods using
labeled dNTP's or ddNTP's. Single-lane sequencing of a specific target DNA
would require distinguishable ddNTP labels. The deoxy- or
dideoxynucleotide triphosphates (dNTP's or ddNTP's) may be labeled with
fluorescent groups or other detectable labels or markers.
Either the Maxam-Gilbert sequencing procedure or Sanger dideoxy sequencing
methods can be used for polynucleotide sequencing reactions. When using
the Maxam Gilbert procedure, either the target sequence itself is
degraded, or alternatively, a sequence complementary to the target
sequence can be synthesized and then degraded.
In the next step, illustrated in FIG. 4(d), the DNA is denatured (hydrogen
bonds between strands are broken) and the labeled chain-terminated
sequencing strands 40L are separated in the gel-containing channels 16 and
18 for detection.
Denaturing can be accomplished using thermal or chemical means to melt the
sequencing strand 40L from the linker oligonucleotide and the target DNA.
A voltage is then applied across the electrodes 30 and 32 to separate the
differently lengthed dideoxy terminated or chemically degraded strands
40L.
FIGS. 5(a) and 5(b) are enlarged views of a strand 40L. FIG. 5(a) shows all
fragments attached, after chain-termination sequencing reactions.
Following denaturing, as seen in FIG. 5(b), the target DNA sequence A
separates from the labeled, dideoxy terminated strand 40L, which also
separates from the linker oligonucleotide 36. Both separated | | |
