U.S. patent application number 10/222298 was filed with the patent office on 2007-04-26 for apparatus and method for sequencing a nucleic acid.
Invention is credited to Joel S. Bader, Jan Berka, Christopher M. Colangelo, Scott B. Dewell, Keith McDade, Jonathan M. Rothberg, John W. Simpson, Michael P. Weiner.
Application Number | 20070092872 10/222298 |
Document ID | / |
Family ID | 25214762 |
Filed Date | 2007-04-26 |
United States Patent
Application |
20070092872 |
Kind Code |
A1 |
Rothberg; Jonathan M. ; et
al. |
April 26, 2007 |
Apparatus and method for sequencing a nucleic acid
Abstract
Disclosed herein are methods and apparatuses for sequencing a
nucleic acid. These methods permit a very large number of
independent sequencing reactions to be arrayed in parallel,
permitting simultaneous sequencing of a very large number
(>10,000) of different oligonucleotides.
Inventors: |
Rothberg; Jonathan M.;
(Guilford, CT) ; Bader; Joel S.; (Stamford,
CT) ; Dewell; Scott B.; (New Haven, CT) ;
McDade; Keith; (Clinton, CT) ; Simpson; John W.;
(Madison, CT) ; Berka; Jan; (New Haven, CT)
; Colangelo; Christopher M.; (Old Lyme, CT) ;
Weiner; Michael P.; (Guildford, CT) |
Correspondence
Address: |
MINTZ LEVIN COHN FERRIS GLOVSKY & POPEO
666 THIRD AVENUE
NEW YORK
NY
10017
US
|
Family ID: |
25214762 |
Appl. No.: |
10/222298 |
Filed: |
August 15, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10104280 |
Mar 21, 2002 |
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10222298 |
Aug 15, 2002 |
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09814338 |
Mar 21, 2001 |
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10104280 |
Mar 21, 2002 |
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09664197 |
Sep 18, 2000 |
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09814338 |
Mar 21, 2001 |
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09398833 |
Sep 16, 1999 |
6274320 |
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09664197 |
Sep 18, 2000 |
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Current U.S.
Class: |
435/6.12 ;
435/287.2; 435/6.1 |
Current CPC
Class: |
B82Y 30/00 20130101;
B01L 2300/0819 20130101; B01L 2300/0877 20130101; B01L 2300/0893
20130101; B01L 2300/0636 20130101; B82Y 15/00 20130101; C12Q 1/6827
20130101; G01N 21/6452 20130101; B01L 2300/0816 20130101; C12Q
1/6869 20130101; B01L 2300/0822 20130101; B01L 3/5027 20130101;
B01L 9/527 20130101; C12Q 1/6837 20130101; C12P 19/34 20130101;
G01N 21/7703 20130101; C12Q 1/6874 20130101; B01L 3/502715
20130101; B01L 2300/0654 20130101; G01N 21/0303 20130101; C12Q
1/6837 20130101; C12Q 2565/519 20130101; C12Q 2533/101 20130101;
C12Q 2531/125 20130101; C12Q 1/6827 20130101; C12Q 2531/125
20130101; C12Q 2535/101 20130101; C12Q 2565/301 20130101; C12Q
1/6837 20130101; C12Q 2531/125 20130101; C12Q 2535/101 20130101;
C12Q 2565/301 20130101; C12Q 1/6837 20130101; C12Q 2565/107
20130101; C12Q 1/6837 20130101; C12Q 2565/107 20130101; C12Q
2525/307 20130101; C12Q 1/6869 20130101; C12Q 2531/125 20130101;
C12Q 2535/101 20130101; C12Q 2565/301 20130101; C12Q 1/6874
20130101; C12Q 2565/107 20130101; C12Q 2525/307 20130101; C12Q
1/6874 20130101; C12Q 2531/125 20130101; C12Q 2535/101 20130101;
C12Q 2565/301 20130101; C12Q 1/6874 20130101; C12Q 2525/307
20130101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Claims
1. A method for sequencing a nucleic acid, the method comprising:
(a) providing one or more nucleic acid anchor primers; (b)
providing a plurality of single-stranded nucleic acid templates
disposed within a plurality of cavities on a planar surface, each
cavity forming an analyte reaction chamber, wherein the reaction
chambers have a center to center spacing of between 5 to 200 .mu.m;
(c) annealing an effective amount of the nucleic acid anchor primer
to at least one of the single-stranded templates to yield a primed
anchor primer-template complex; (d) combining the primed anchor
primer-template complex with a polymerase to form an extended
anchor primer covalently linked to multiple copies of a nucleic
acid complementary to the nucleic acid template; (e) annealing an
effective amount of a sequencing primer to one or more copies of
said covalently linked complementary nucleic acid; (f) extending
the sequencing primer with a polymerase and a predetermined
nucleotide triphosphate to yield a sequencing product and, if the
predetermined nucleotide triphosphate is incorporated onto the 3'
end of said sequencing primer, a sequencing reaction byproduct; and
(g) identifying the sequencing reaction byproduct, thereby
determining the sequence of the nucleic acid.
2. The method of claim 1 wherein each single stranded nucleic acid
contains at least 100 copies of a nucleic acid sequence, each copy
covalently linked end to end.
3. The method of claim 1 wherein said single stranded nucleic acid
template is a circular single stranded nucleic acid template.
4. The method of claim 1 wherein each reaction chamber has a width
in at least one dimension of between 0.3 .mu.m and 100 .mu.m.
5. The method of claim 1 wherein each reaction chamber has a width
in at least one dimension of between 0.3 .mu.m and 20 .mu.m.
6. The method of claim 1 wherein each reaction chamber has a width
in at least one dimension of between 0.3 .mu.m and 10 .mu.m.
7. The method of claim 1 wherein each reaction chamber has a width
in at least one dimension of between 20 .mu.m and 70 .mu.m.
8. The method of claim 1 wherein the cavities number greater than
400,000.
9. The method of claim 1 wherein the cavities number between
400,000 and 20,000,000.
10. The method of claim 1 wherein the cavities number between
1,000,000 and 16,000,000.
11. The method of claim 1 wherein the center to center spacing is
between 10 to 150 .mu.m.
12. The method of claim 1 wherein the center to center spacing is
between 50 to 100 .mu.m.
13. The method of claim 1 wherein each cavity has a depth of
between 10 .mu.m and 100 .mu.m.
14. The method of claim 1 wherein each cavity has a depth that is
between 0.25 and 5 times the size of the width of the cavity.
15. The method of claim 1 wherein each cavity has a depth that is
between 0.3 and 1 times the size of the width of the cavity.
16. The method of claim 1 wherein the nucleic acid sequence is
further amplified to produce multiple copies of said nucleic acid
sequence after being disposed in the reaction chamber.
17. The method of claim 16 wherein the nucleic acid sequence is
amplified using an amplification technology selected from the group
consisting of polymerase chain reaction, ligase chain reaction and
isothermal DNA amplification.
18. The method of claim 1 wherein the single stranded nucleic acid
is immobilized in the reaction chamber.
19. The method of claim 1 wherein the single stranded nucleic acid
is immobilized on one or more mobile solid supports disposed in the
reaction chamber.
20. A method for sequencing a nucleic acid, the method comprising:
(a) providing at least one nucleic acid anchor primer; (b)
providing a plurality of single-stranded nucleic acid templates in
an array having at least 400,000 discrete reaction sites; (c)
annealing a first amount of the nucleic acid anchor primer to at
least one of the single-stranded templates to yield a primed anchor
primer-template complex; (d) combining the primed anchor
primer-template complex with a polymerase to form an extended
anchor primer covalently linked to multiple copies of a nucleic
acid complementary to the nucleic acid template; (e) annealing a
second amount of a sequencing primer to one or more copies of the
covalently linked complementary nucleic acid; (f) extending the
sequencing primer with a polymerase and a predetermined nucleotide
triphosphate to yield a sequencing product and, when the
predetermined nucleotide triphosphate is incorporated onto the 3'
end of the sequencing primer, to yield a sequencing reaction
byproduct; and (g) identifying the sequencing reaction byproduct,
thereby determining the sequence of the nucleic acid at each
reaction site that contains a nucleic acid template.
21. The method of claim 20 wherein said single-stranded nucleic
acid template is a circular single-stranded nucleic acid
template.
22. The method of claim 20 wherein the anchor primer is linked to a
particle.
23. The method of claim 20 wherein the anchor primer is linked to
the particle prior to formation of the extended anchor primer.
24. The method of claim 20 wherein the anchor primer is linked to
the particle after formation of the extended anchor primer.
25. The method of claim 20 wherein the sequencing reaction
byproduct is PPi and a coupled sulfurylase/luciferase reaction is
used to generate light for detection.
26. The method of claim 25 wherein either or both of the
sulfurylase and luciferase are immobilized on one or more mobile
solid supports disposed at each reaction site.
27. A method of determining the base sequence of a plurality of
nucleotides on an array, the method comprising: (a) providing a
plurality of sample DNAs, each disposed within a plurality of
cavities on a planar surface, each cavity forming an analyte
reaction chamber, wherein the reaction chambers have a center to
center spacing of between 5 to 200 .mu.m; (b) adding an activated
nucleotide 5'-triphosphate precursor of one known nitrogenous base
to a reaction mixture in each reaction chamber, each reaction
mixture comprising a template-directed nucleotide polymerase and a
single-stranded polynucleotide template hybridized to a
complementary oligonucleotide primer strand at least one nucleotide
residue shorter than the templates to form at least one unpaired
nucleotide residue in each template at the 3'-end of the primer
strand, under reaction conditions which allow incorporation of the
activated nucleoside 5'-triphosphate precursor onto the 3'-end of
the primer strands, provided the nitrogenous base of the activated
nucleoside 5'-triphosphate precursor is complementary to the
nitrogenous base of the unpaired nucleotide residue of the
templates; (c) detecting whether or not the nucleoside
5'-triphosphate precursor was incorporated into the primer strands
in which incorporation of the nucleoside 5'-triphosphate precursor
indicates that the unpaired nucleotide residue of the template has
a nitrogenous base composition that is complementary to that of the
incorporated nucleoside 5'-triphosphate precursor; (d) sequentially
repeating steps (b) and (c), wherein each sequential repetition
adds and, detects the incorporation of one type of activated
nucleoside 5'-triphosphate precursor of known nitrogenous base
composition; and (e) determining the base sequence of the unpaired
nucleotide residues of the template in each reaction chamber from
the sequence of incorporation of said nucleoside precursors.
28. A method for determining the nucleic acid sequence in a
template nucleic acid polymer, comprising: (a) introducing a
plurality of template nucleic acid polymers into a plurality of
cavities on a planar surface, each cavity forming an analyte
reaction chamber, wherein the reaction chambers have a center to
center spacing of between 5 to 200 .mu.m, each reaction chamber
having a polymerization environment in which the nucleic acid
polymer will act as a template polymer for the synthesis of a
complementary nucleic acid polymer when nucleotides are added; (b)
successively providing to the polymerization environment a series
of feedstocks, each feedstock comprising a nucleotide selected from
among the nucleotides from which the complementary nucleic acid
polymer will be formed, such that if the nucleotide in the
feedstock is complementary to the next nucleotide in the template
polymer to be sequenced said nucleotide will be incorporated into
the complementary polymer and inorganic pyrophosphate will be
released; and (c) detecting the formation of inorganic
pyrophosphate to determine the identify of each nucleotide in the
complementary polymer and thus the sequence of the template
polymer.
29. A method of identifying the base in a target position in a DNA
sequence of sample DNA, wherein: (a) disposing sample DNA within a
plurality of cavities on a planar surface, each cavity forming an
analyte reaction chamber, wherein the reaction chambers have a
center to center spacing of between 5 to 200 .mu.m, said DNA being
rendered single stranded either before or after being disposed in
the reaction chambers, (b) providing an extension primer which
hybridizes to said immobilized single-stranded DNA at a position
immediately adjacent to said target position; (c) subjecting said
immobilized single-stranded DNA to a polymerase reaction in the
presence of a predetermined nucleotide triphosphate, wherein if the
predetermined nucleotide triphosphate is incorporated onto the 3'
end of said sequencing primer then a sequencing reaction byproduct
is formed; and (d) identifying the sequencing reaction byproduct,
thereby determining the nucleotide complementary to the base at
said target position.
30. A method of identifying a base at a target position in a sample
DNA sequence comprising: (a) providing sample DNA disposed within a
plurality of cavities on a planar surface, each cavity forming an
analyte reaction chamber, wherein the reaction chambers have a
center to center spacing of between 5 to 200 .mu.m, said DNA being
rendered single stranded either before or after being disposed in
the reaction chambers; (b) providing an extension primer which
hybridizes to the sample DNA immediately adjacent to the target
position; (c) subjecting the sample DNA sequence and the extension
primer to a polymerase reaction in the presence of a nucleotide
triphosphate whereby the nucleotide triphosphate will only become
incorporated and release pyrophosphate (PPi) if it is complementary
to the base in the target position, said nucleotide triphosphate
being added either to separate aliquots of sample-primer mixture or
successively to the same sample-primer mixture; and (d) detecting
the release of PPi to indicate which nucleotide is
incorporated.
31. A method of identifying a base at a target position in a
single-stranded sample DNA sequence, the method comprising: (a)
providing an extension primer which hybridizes to sample DNA
immediately adjacent to the target position, said sample DNA
disposed within a plurality of cavities on a planar surface, each
cavity forming an analyte reaction chamber, wherein the reaction
chambers have a center to center spacing of between 5 to 200 um,
said DNA being rendered single stranded either before or after
being disposed in the reaction chambers; (b) subjecting the sample
DNA and extension primer to a polymerase reaction in the presence
of a predetermined deoxynucleotide or dideoxynucleotide whereby the
deoxynucleotide or dideoxynucleotide will only become incorporated
and release pyrophosphate (PPi) if it is complementary to the base
in the target position, said predetermined deoxynucleotides or
dideoxynucleotides being added either to separate aliquots of
sample-primer mixture or successively to the same sample-primer
mixture; and (c) detecting any release of PPi enzymatically to
indicate which deoxynucleotide or dideoxynucleotide is
incorporated; characterized in that, the PPi-detection enzyme(s)
are included in the polymerase reaction step and in that in place
of deoxy- or dideoxy adenosine triphosphate (ATP) a dATP or ddATP
analogue is used which is capable of acting as a substrate for a
polymerase but incapable of acting as a substrate for a said
PPi-detection enzyme.
32. A method of determining the base sequence of a plurality of
nucleotides on an array, the method comprising: (a) providing a
plurality of sample DNAs, each disposed within a plurality of
cavities on a planar surface, each cavity forming an analyte
reaction chamber, wherein the reaction chambers have a center to
center spacing of between 5 to 200 .mu.m; (b) detecting the light
level emitted from a plurality of reaction sites on respective
portions of an optically sensitive device; (c) converting the light
impinging upon each of said portions of said optically sensitive
device into an electrical signal which is distinguishable from the
signals from all of said other regions; (d) determining a light
intensity for each of said discrete regions from the corresponding
electrical signal; and (e) recording the variations of said
electrical signals with time.
33. Method for sequencing a nucleic acid, the method comprising:
(a) providing one or more nucleic acid anchor primers; (b)
providing a plurality of single-stranded nucleic acid templates
disposed within a plurality of cavities on a planar surface, each
cavity forming an analyte reaction chamber, wherein the reaction
chambers have a center to center spacing of between 5 to 200 .mu.m;
(c) detecting the light level emitted from a plurality of reaction
sites on respective portions of an optically sensitive device; (d)
converting the light impinging upon each of said portions of said
optically sensitive device into an electrical signal which is
distinguishable from the signals from all of said other regions;
(e) determining a light intensity for each of said discrete regions
from the corresponding electrical signal; and (f) recording the
variations of said electrical signals with time.
34. A method for sequencing a nucleic acid, the method comprising:
(a) providing at least one nucleic acid anchor primer; (b)
providing a plurality of single-stranded nucleic acid templates in
an array having at least 400,000 discrete reaction sites; (c)
detecting the light level emitted from a plurality of reaction
sites on respective portions of an optically sensitive device; (d)
converting the light impinging upon each of said portions of said
optically sensitive device into an electrical signal which is
distinguishable from the signals from all of said other regions;
(e) determining a light intensity for each of said discrete regions
from the corresponding electrical signal; and (f) recording the
variations of said electrical signals with time.
Description
[0001] RELATED APPLICATIONS
[0002] This application claims the benefit of priority to U.S. Ser.
No. 10/104,280 filed Mar. 21, 2002; which is a CIP of 09/814,338
filed Mar. 21, 2001; which is a CIP of U.S. Ser. No. 09/664,197
filed Sep. 18, 2000; which is a CIP of U.S. Ser. No. 09/398,833
filed Sep. 16, 1999, now U.S. Pat. No. 6,274,320. Each of the above
referenced patent and patent applications are incorporated herein
by reference in their entireties.
FIELD OF THE INVENTION
[0003] The invention relates to apparatus and methods for
determining the sequence of a nucleic acid.
BACKGROUND OF THE INVENTION
[0004] Many diseases are associated with particular DNA sequences.
The DNA sequences are often referred to as DNA sequence
polymorphisms to indicate that the DNA sequence associated with a
diseased state differs from the corresponding DNA sequence in
non-afflicted individuals. DNA sequence polymorphisms can include,
e.g., insertions, deletions, or substitutions of nucleotides in one
sequence relative to a second sequence. An example of a particular
DNA sequence polymorphism is 5'-ATCG-3, relative to the sequence
5'-ATGG-3' at a particular location in the human genome. The first
nucleotide `G` in the latter sequence has been replaced by the
nucleotide `C` in the former sequence. The former sequence is
associated with a particular disease state, whereas the latter
sequence is found in individuals not suffering from the disease.
Thus, the presence of the nucleotide sequence `5-ATCG-3` indicates
the individual has the particular disease. This particular type of
sequence polymorphism is known as a single-nucleotide polymorphism,
or SNP, because the sequence difference is due to a change in one
nucleotide.
[0005] Techniques which enable the rapid detection of as little as
a single DNA base change are therefore important methodologies for
use in genetic analysis. Because the size of the human genome is
large, on the order of 3 billion base pairs, techniques for
identifying polymorphisms must be sensitive enough to specifically
identify the sequence containing the polymorphism in a potentially
large population of nucleic acids.
[0006] Typically a DNA sequence polymorphism analysis is performed
by isolating DNA from an individual, manipulating the isolated DNA,
e.g., by digesting the DNA with restriction enzymes and/or
amplifying a subset of sequences in the isolated DNA. The
manipulated DNA is then examined further to determine if a
particular sequence is present.
[0007] Commonly used procedures for analyzing the DNA include
electrophoresis. Common applications of electrophoresis include
agarose or polyacrylamide gel electrophoresis. DNA sequences are
inserted, or loaded, on the gels and subjected to an electric
field. Because DNA carries a uniform negative charge, DNA will
migrate through the gel based on properties including sequence
length, three-dimensional conformation and interactions with the
gel matrix upon application of the electrical field. In most
applications, smaller DNA molecules will migrate more rapidly
through the gel than larger fragments. After electrophoresis has
been continued for a sufficient length of time, the DNA molecules
in the initial population of DNA sequences will have been separated
according to their relative sizes.
[0008] Particular DNA molecules can then be detected using a
variety of detection methodologies. For some applications,
particular DNA sequences are identified by the presence of
detectable tags, such as radioactive labels, attached to specific
DNA molecules.
[0009] Electrophoretic-based separation analyses can be less
desirable for applications in which it is desirable to rapidly,
economically, and accurately analyze a large number of nucleic acid
samples for particular sequence polymorphisms. For example,
electrophoretic-based analysis can require a large amount of input
DNA. In addition, processing the large number of samples required
for electrophoretic-based nucleic acid based analyses can be labor
intensive. Furthermore, these techniques can require samples of
identical DNA molecules, which must be created prior to
electrophoresis at costs that can be considerable.
[0010] Recently, automated electrophoresis systems have become
available. However, electrophoresis can be ill suited for
applications such as clinical sequencing, where relatively
cost-effective units with high throughput are needed. Thus, the
need for non-electrophoretic methods for sequencing is great. For
many applications, electrophoresis is used in conjunction with DNA
sequence analysis.
[0011] Several alternatives to electrophoretic-based sequencing
have been described. These include scanning tunnel electron
microscopy, sequencing by hybridization, and single molecule
detection methods.
[0012] Another alternative to electrophoretic-based separation
analysis is solid substrate-based nucleic acid analyses. These
methods typically rely upon the use of large numbers of nucleic
acid probes affixed to different locations on a solid support.
These solid supports can include, e.g., glass surfaces, plastic
microtiter plates, plastic sheets, thin polymers, or
semi-conductors. The probes can be, e.g., adsorbed or covalently
attached to the support, or can be microencapsulated or otherwise
entrapped within a substrate matrix, membrane, or film.
[0013] Substrate-based nucleic acid analyses can include applying a
sample nucleic acid known or suspected of containing a particular
sequence polymorphism to an array of probes attached to the solid
substrate. The nucleic acids in the population are allowed to
hybridize to complementary sequences attached to the substrate, if
present. Hybridizing nucleic acid sequences are then detected in a
detection step.
[0014] Solid support matrix-based hybridization and sequencing
methodologies can require a high sample-DNA concentration and can
be hampered by the relatively slow hybridization kinetics of
nucleic acid samples with immobilized oligonucleotide probes.
Often, only a small amount of template DNA is available, and it can
be desirable to have high concentrations of the target nucleic acid
sequence. Thus, substrate based detection analyses often include a
step in which copies of the target nucleic acid, or a subset of
sequences in the target nucleic acid, is amplified. Methods based
on the Polymerase Chain Reaction (PCR), e.g., can increase a small
number of probe targets by several orders of magnitude in solution.
However, PCR can be difficult to incorporate into a solid-phase
approach because the amplified DNA is not immobilized onto the
surface of the solid support matrix.
[0015] Solid-phase based detection of sequence polymorphisms has
been described. An example is a "mini-sequencing" protocol based
upon a solid phase principle described by Hultman, et al., 1988.
Nucl. Acid. Res. 17: 4937-4946; Syvanen, et al., 1990. Genomics 8:
684-692. In this study, the incorporation of a radiolabeled
nucleotide was measured and used for analysis of a three-allelic
polymorphism of the human apolipoprotein E gene. However, such
radioactive methods are not well suited for routine clinical
applications, and hence the development of a simple, highly
sensitive non-radioactive method for rapid DNA sequence analysis
has also been of great interest.
SUMMARY OF THE INVENTION
[0016] The invention is based in part on the use of arrays for
determining the sequences of nucleic acids.
[0017] Accordingly, in one aspect, the invention involves an array
including a planar surface with a plurality of reaction chambers
disposed thereon, wherein the reaction chambers have a center to
center spacing of between 5 to 200 .mu.m and each chamber has a
width in at least one dimension of between 0.3 .mu.m and 100 .mu.m.
In some embodiments, the array is a planar surface with a plurality
of cavities thereon, where each cavity forms an analyte reaction
chamber. In a preferred embodiment, the array is fashioned from a
sliced fiber optic bundle (i.e., a bundle of fused fiber optic
cables) and the reaction chambers are formed by etching one surface
of the fiber optic reactor array ("FORA"). The cavities can also be
formed in the substrate via etching, molding or micromachining.
[0018] Specifically, each reaction chamber in the array typically
has a width in at least one dimension of between 0.3 .mu.m and 100
.mu.m, preferably between 0.3 .mu.m and 20 .mu.m, mst preferably
between 0.3 .mu.m and 10 .mu.m. In a separate embodiment, we
contemplate larger reaction chambers, preferably having a width in
at least one dimension of between 20 .mu.m and 70 .mu.m.
[0019] The array typically contains more than 1,000 reaction
chambers, preferably more than 400,000, more preferably between
400,000 and 20,000,000, and most preferably between 1,000,000 and
16,000,000 cavities or reaction chambers. The shape of each cavity
is frequently substantially hexagonal, but the cavities can also be
cylindrical. In some embodiments, each cavity has a smooth wall
surface, however, we contemplate that each cavity may also have at
least one irregular wall surface. The bottom of each of the
cavities can be planar or concave.
[0020] The array is typically constructed to have cavities or
reaction chambers with a center-to-center spacing between 10 to 150
.mu.m, preferably between 50 to 100 .mu.m.
[0021] Each cavity or reaction chamber typically has a depth of
between 10 .mu.m and 100 .mu.m; alternatively, the depth is between
0.25 and 5 times the size of the width of the cavity, preferably
between 0.3 and 1 times the size of the width of the cavity.
[0022] In one embodiment, the arrays described herein typically
include a planar top surface and a planar bottom surface, which is
optically conductive such that optical signals from the reaction
chambers can be detected through the bottom planar surface. In
these arrays, typically the distance between the top surface and
the bottom surface is no greater than 10 cm, preferably no greater
than 3 cm, most preferably no greater than 2 cm, and usually
between 0.5 mm to 5 mm.
[0023] In one embodiment, each cavity of the array contains
reagents for analyzing a nucleic acid or protein. The array can
also include a second surface spaced apart from the planar array
and in opposing contact therewith such that a flow chamber is
formed over the array.
[0024] In another aspect, the invention involves an array means for
carrying out separate parallel common reactions in an aqueous
environment, wherein the array means includes a substrate having at
least 1,000 discrete reaction chambers. These chambers contain a
starting material that is capable of reacting with a reagent. Each
of the reaction chambers are dimensioned such that when one or more
fluids containing at least one reagent is delivered into each
reaction chamber, the diffusion time for the reagent to diffuse out
of the well exceeds the time required for the starting material to
react with the reagent to form a product. The reaction chambers can
be formed by generating a plurality of cavities on the substrate,
or by generating discrete patches on a planar surface, the patches
having a different surface chemistry than the surrounding planar
surface.
[0025] In one embodiment, each cavity or reaction chamber of the
array contains reagents for analyzing a nucleic acid or protein.
Typically those reaction chambers that contain a nucleic acid (not
all reaction chambers in the array are required to) contain only a
single species of nucleic acid (i.e., a single sequence that is of
interest). There may be a single copy of this species of nucleic
acid in any particular reaction chamber, or they may be multiple
copies. It is generally preferred that a reaction chamber contain
at least 100 copies of a nucleic acid sequence, preferably at least
100,000 copies, and most preferably between 100,000 to 1,000,000
copies of the nucleic acid. In one embodiment the nucleic acid
species is amplified to provide the desired number of copies using
PCR, RCA, ligase chain reaction, other isothermal amplification, or
other conventional means of nucleic acid amplification. In one
embodimant, the nucleic acid is single stranded. In other
embodiments the single stranded DNA is a concatamer with each copy
covalently linked end to end.
[0026] The nucleic acid may be immobilized in the reaction chamber,
either by attachment to the chamber itself or by attachment to a
mobile solid support that is delivered to the chamber. A bioactive
agent could be delivered to the array, by dispersing over the array
a plurality of mobile solid supports, each mobile solid support
having at least one reagent immobilized thereon, wherein the
reagent is suitable for use in a nucleic acid sequencing
reaction.
[0027] The array can also include a population of mobile solid
supports disposed in the reaction chambers, each mobile solid
support having one or more bioactive agents (such as a nucleic acid
or a sequencing enzyme) attached thereto. The diameter of each
mobile solid support can vary, we prefer the diameter of tie mobile
solid support to be between 0.01 to 0.1 times the width of each
cavity. Not every reaction chamber need contain one or more mobile
solid supports. There are three contemplated embodiments; one where
at least 5% to 20% of of the reaction chambers can have a mobile
solid support having at least one reagent immobilized thereon; a
second embodiment where 20% to 60% of the reaction chambers can
have a mobile solid support having at least one reagent immobilized
thereon; and a third embodiment where 50% to 100% of the reaction
chambers can have a mobile solid support having at least one
reagent immobilized thereon.
[0028] The mobile solid support typically has at least one reagent
immobilized thereon. For the embodiments relating to pyrosequencing
reactions or more generally to ATP detection, the reagent may be a
polypeptide with sulfurylase or luciferase activity, or both. The
mobile solid supports can be used in methods for dispersing over
the array a plurality of mobile solid supports having one or more
nucleic sequences or proteins or enzymes immobilized thereon.
[0029] In another aspect, the invention involves an apparatus for
simultaneously monitoring the array of reaction chambers for light
generation, indicating that a reaction is taking place at a
particular site. In this embodiment, the reaction chambers are
sensors, adapted to contain analytes and an enzymatic or
fluorescent means for generating light in the reaction chambers. In
this embodiment of the invention, the sensor is suitable for use in
a biochemical or cell-based assay. The apparatus also includes an
optically sensitive device arranged so that in use the light from a
particular reaction chamber would impinge upon a particular
predetermined region of the optically sensitive device, as well as
means for determining the light level impinging upon each of the
predetermined regions and means to record the variation of the
light level with time for each of the reaction chamber.
[0030] In one specific embodiment, the instrument includes a light
detection means having a light capture means and a second fiber
optic bundle for transmitting light to the light detecting means.
We contemplate one light capture means to be a CCD camera. The
second fiber optic bundle is typically in optical contact with the
array, such that light generated in an individual reaction chamber
is captured by a separate fiber or groups of separate fibers of the
second fiber optic bundle for transmission to the light capture
means.
[0031] The above arrays may be used for carrying out separate
parallel common reactions in an aqueous environment. The method
includes delivering a fluid containing at least one reagent to the
described arrays, wherein certain reaction chambers (not
necessarily all) on the array contain a starting material that is
capable of reacting with the reagent. Each of the reaction chambers
is dimensioned such that when the fluid is delivered into each
reaction chamber, the diffusion time for the reagent to diffuse out
of the well exceeds the time required for the starting material to
react with the reagent to form a product. The method also includes
washing the fluid from the array in the time period after the
starting material has reacted with the reagent to form a product in
each reaction chamber but before the reagent delivered to any one
reaction chamber has diffused out of that reaction chamber into any
other reaction chamber. In one embodiment, the product formed in
any one reaction chamber is independent of the product formed in
any other reaction chamber, but is generated using one or more
common reagents. The starting material can be a nucleic acid
sequence and at least one reagent in the fluid is a nucleotide or
nucleotide analog. The fluid can additionally have a polymerase
capable of reacting the nucleic acid sequence and the nucleotide or
nucleotide analog. The steps of the method can be repeated
sequentially.
[0032] The apparatus includes a novel reagent delivery cuvette
adapted for use with the arrays described herein, to provide fluid
reagents to the array, and a reagent delivery means in
communication with the reagent delivery cuvette.
[0033] The disclosures of one or more embodiments of the invention
are set forth in the accompanying description below. Although any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
Other features, objects, and advantages of the invention will be
apparent from the description and from the claims. In the
specification and the appended claims, the singular forms include
plural referents unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Unless
expressly stated otherwise, the techniques employed or contemplated
herein are standard methodologies well known to one of ordinary
skill in the art. The examples of embodiments are for illustration
purposes only. All patents and publications cited in this
specification are incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIGS. 1A-D are schematic illustrations of rolling
circle-based amplification using an anchor primer.
[0035] FIG. 2 is a drawing of a sequencing apparatus according to
the present invention.
[0036] FIG. 3 is a drawing of a perfusion chamber according to the
present invention.
[0037] FIG. 4 is a drawing of a cavitated fiber optic terminus of
the present invention.
[0038] FIG. 5 is a tracing of a sequence output of a concatemeric
template generated using rolling circle amplification.
[0039] FIG. 6 is a micrograph of a Fiber Optic Reactor Array
(FORA).
[0040] FIG. 7 is a schematic illustration for the the preparation
of a carpeted FORA.
[0041] FIG. 8 is a micrograph for single well DNA delivery.
[0042] FIG. 9 is a schematic illustration of the Flow Chamber and
FORA.
[0043] FIG. 10 is a diagram of the analytical instrument of the
present invention.
[0044] FIG. 11 is a schematic illustration of microscopic parallel
sequencing reactions within a FORA.
[0045] FIG. 12 is a micrograph of single well reactions.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The methods and apparatuses described herein allow for the
determination of nucleic acid sequence information without the need
for first cloning a nucleic acid. In addition, the method is highly
sensitive and can be used to determine the nucleotide sequence of a
template nucleic acid, which is present in only a few copies in a
starting population of nucleic acids. Further, the method can be
used to determine simultaneously the sequences of a large number of
nucleic acids.
[0047] The methods and apparatuses described are generally useful
for any application in which the identification of any particular
nucleic acid sequence is desired. For example, the methods allow
for identification of single nucleotide polymorphisms (SNPs),
haplotypes involving multiple SNPs or other polymorphisms on a
single chromosome, and transcript profiling. Other uses include
sequencing of artificial DNA constructs to confirm or elicit their
primary sequence, or to identify specific mutant clones from random
mutagenesis screens, as well as to obtain the sequence of cDNA from
single cells, whole tissues or organisms from any developmental
stage or environmental circumstance in order to determine the gene
expression profile from that specimen. In addition, the methods
allow for the sequencing of PCR products and/or cloned DNA
fragments of any size isolated from any source.
[0048] The methods described herein include a sample preparation
process that results in a solid or a mobile solid substrate array
containing a plurality of anchor primers covalently linked to a
nucleic acid containing one or more copies complementary to a
target nucleic acid. Formation of the covalently linked anchor
primer and one or more copies of the target nucleic acid preferably
occurs by annealing the anchor primer to a complementary region of
a circular nucleic acid, and then extending the annealed anchor
primer with a polymerase to result in formation of a nucleic acid
containing one or more copies of a sequence complementary to the
circular nucleic acid.
[0049] Attachment of the anchor primer to a solid or mobile solid
substrate can occur before, during, or subsequent to extension of
the annealed anchor primer. Thus, in one embodiment, one or more
anchor primers are linked to the solid or a mobile solid substrate,
after which the anchor primer is annealed to a target nucleic acid
and extended in the presence of a polymerase. Alternatively, in a
second embodiment, an anchor primer is first annealed to a target
nucleic acid, and a 3'OH terminus of the annealed anchor primer is
extended with a polymerase. The extended anchor primer is then
linked to the solid or mobile solid substrate. By varying the
sequence of anchor primers, it is possible to specifically amplify
distinct target nucleic acids present in a population of nucleic
acids.
[0050] Sequences in the target nucleic acid can be identified in a
number of ways. Preferably, a sequencing primer is annealed to the
amplified nucleic acid and used to generate a sequencing product.
The nucleotide sequence of the sequence product is then determined,
thereby allowing for the determination of the nucleic acid.
Similarly, in one embodiment, the template nucleic acid is
amplified prior to its attachment to the bead or other mobile solid
support. In other embodiments, the template nucleic acid is
attached to the bead prior to its amplification.
[0051] The methods of the present invention can be also used for
the sequencing of DNA fragments generated by analytical techniques
that probe higher order DNA structure by their differential
sensitivity to enzymes, radiation or chemical treatment (e.g.,
partial DNase treatment of chromatin), or for the determination of
the methylation status of DNA by comparing sequence generated from
a given tissue with or without prior treatment with chemicals that
convert methyl-cytosine to thymidine (or other nucleotide) as the
effective base recognized by the polymerase. Further, the methods
of the present invention can be used to assay cellular physiology
changes occurring during development or senescence at the level of
primary sequence.
[0052] The invention also provides methods of preparing nucleic
acid sequences for subsequent analysis, e.g., sequencing.
I. Apparatus for Sequencing Nucleic Acids
[0053] This invention provides an apparatus for sequencing nucleic
acids, which generally comprises one or more reaction chambers for
conducting a sequencing reaction, means for delivering reactants to
and from the reaction chamber(s), and means for detecting a
sequencing reaction event. In another embodiment, the apparatus
includes a reagent delivery cuvette containing a plurality of
cavities on a planar surface. In a preferred embodiment, the
apparatus is connected to at least one computer for controlling the
individual components of the apparatus and for storing and/or
analyzing the information obtained from detection of the sequence
reaction event.
[0054] The invention also provides one or more reaction chambers
are arranged in the form of an array on an inert substrate
material, also referred to herein as a "solid support", that allows
for combination of the reactants in a sequencing reaction in a
defined space and for detection of the sequencing reaction event.
Thus, as used herein, the terms "reaction chamber" or "analyte
reaction chamber" refer to a localized area on the substrate
material that facilitates interaction of reactants, e.g., in a
nucleic acid sequencing reaction. As discussed more fully below,
the sequencing reactions contemplated by the invention preferably
occur on numerous individual nucleic acid samples in tandem, in
particular simultaneously sequencing numerous nucleic acid samples
derived from genomic and chromosomal DNA. The apparatus of the
invention therefore preferably comprises an array having a
sufficient number of reaction chambers to carry out such numerous
individual sequencing reactions. In one embodiment, the array
comprises at least 1,000 reaction chambers. In another embodiment,
the array comprises greater than 400,000 reaction chambers,
preferably between 400,000 and 20,000,000 reaction chambers. In a
more preferred embodiment, the array comprises between 1,000,000
and 16,000,000 reaction chambers.
[0055] The reaction chambers on the array typically take the form
of a cavity or well in the substrate material, having a width and
depth, into which reactants can be deposited. One or more of the
reactants typically are bound to the substrate material in the
reaction chamber and the remainder of the reactants are in a medium
which facilitates the reaction and which flows through the reaction
chamber. When formed as cavities or wells, the chambers are
preferably of sufficient dimension and order to allow for (i) the
introduction of the necessary reactants into the chambers, (ii)
reactions to take place within the chamber and (iii) inhibition of
mixing of reactants between chambers. The shape of the well or
cavity is preferably circular or cylindrical, but can be multisided
so as to approximate a circular or cylindrical shape. In another
embodiment, the shape of the well or cavity is substantially
hexagonal. The cavity can have a smooth wall surface. In an
additional embodiment, the cavity can have at least one irregular
wall surface. The cavities can have a planar bottom or a concave
bottom. The reaction chambers can be spaced between 5 .mu.m and 200
.mu.m apart. Spacing is determined by measuring the
center-to-center distance between two adjacent reaction chambers.
Typically, the reaction chambers can be spaced between 10 .mu.m and
150 .mu.m apart, preferably between 50 .mu.m and 100 .mu.m apart.
In one embodiment, the reaction chambers have a width in one
dimension of between 0.3 .mu.m and 100 .mu.m. The reaction chambers
can have a width in one dimension of between 0.3 .mu.m and 20
.mu.m, preferably between 0.3 .mu.m and 10 .mu.m, and most
preferably about 6 .mu.m. In another embodiment, the reaction
chambers have a width of between 20 .mu.m and 70 .mu.m Ultimately
the width of the chamber may be dependant on whether the nucleic
acid samples require amplification. If no amplification is
necessary, then smaller, e.g., 0.3 .mu.m is preferred. If
amplification is necessary, then larger, e.g., 6 .mu.m is
preferred. The depth of the reaction chambers are preferably
between 10 .mu.m and 100 .mu.m. Alternatively, the reaction
chambers may have a depth that is between 0.25 and 5 times the
width in one dimension of the reaction chamber or, in another
embodiment, between 0.3 and 1 times the width in one dimension of
the reaction chamber.
[0056] In another aspect, the invention involves an apparatus for
determining the nucleic acid sequence in a template nucleic acid
polymer. The apparatus includes an array having a plurality of
cavities on a planar surface. Each cavity forms an analyte reaction
chamber, wherein the reaction chambers have a center-to-center
spacing of between 5 to 200 .mu.m. It also includes a nucleic acid
delivery means for introducing a template nucleic acid polymers
into the reaction chambers; and a nucleic acid delivery means to
deliver reagents to the reaction chambers to create a
polymerization environment in which the nucleic acid polymers will
act as a template polymers for the synthesis of complementary
nucleic acid polymers when nucleotides are added. The apparatus
also includes a reagent delivery means for successively providing
to the polymerization environment a series of feedstocks, each
feedstock comprising a nucleotide selected from among the
nucleotides from which the complementary nucleic acid polymer will
be formed, such that if the nucleotide in the feedstock is
complementary to the next nucleotide in the template polymer to be
sequenced the nucleotide will be incorporated into the
complementary polymer and inorganic pyrophosphate will be released.
It also includes a detection means for detecting the formation of
inorganic pyrophosphate enzymatically; and a data processing means
to determine the identity of each nucleotide in the complementary
polymers and thus the sequence of the template polymers.
[0057] In another aspect, the invention involves an apparatus for
determining the base sequence of a plurality of nucleotides on an
array. The apparatus includes a reagent cuvette containing a
plurality of cavities on a planar surface. Each cavity forms an
analyte reaction chamber, wherein the reaction chambers have a
center-to-center spacing of between 5 to 200 .mu.m. The apparatus
also includes a reagent delivery means for adding an activated
nucleotide 5'-triphosphate precursor of one known nitrogenous base
to a reaction mixture in each reaction chamber. Each reaction
mixture has a template-directed nucleotide polymerase and a
single-stranded polynucleotide template hybridized to a
complementary oligonucleotide primer strand at least one nucleotide
residue shorter than the templates to form at least one unpaired
nucleotide residue in each template at the 3'-end of the primer
strand, under reaction conditions which allow incorporation of the
activated nucleoside 5'-triphosphate precursor onto the 3'-end of
the primer strands, provided the nitrogenous base of the activated
nucleoside 5'-triphosphate precursor is complementary to the
nitrogenous base of the unpaired nucleotide residue of the
templates. The apparatus also includes a detection means for
detecting whether or not the nucleoside 5'-triphosphate precursor
was incorporated into the primer strands in which incorporation of
the nucleoside 5'-triphosphate precursor indicates that the
unpaired nucleotide residue of the template has a nitrogenous base
composition that is complementary to that of the incorporated
nucleoside 5'-triphosphate precursor. The apparatus also includes a
means for sequentially repeating the second and third steps wherein
each sequential repetition adds and, detects the incorporation of
one type of activated nucleoside 5'-triphosphate precursor of known
nitrogenous base composition. The apparatus also includes a data
processing means for determining the base sequence of the unpaired
nucleotide residues of the template in each reaction chamber from
the sequence of incorporation of the nucleoside precursors.
Solid Support Material
[0058] Any material can be used as the solid support material, as
long as the surface allows for stable attachment of the primers and
detection of nucleic acid sequences. The solid support material can
be planar or can be cavitated, e.g., in a cavitated terminus of a
fiber optic or in a microwell etched, molded, or otherwise
micromachined into the planar surface, e.g. using techniques
commonly used in the construction of microelectromechanical
systems. See e.g., Rai-Choudhury HANDBOOK OF MICROLITHOGRAPHY,
MICROMACHINING, AND MICROFABRICATION, VOLUME I: MICROLITHOGRAPHY,
Volume PM 39, SPIE Press (1997); Madou, CRC Press (1997), Aoki,
Biotech. Histochem. 67: 98-9 (1992); Kane et al., Biomaterials, 20:
2363-76 (1999); Deng et al., Anal. Chem. 72:3176-80 (2000); Zhu et
al., Nat. Genet. 26:283-9 (2000). In some embodiments, the solid
support is optically transparent, e.g., glass.
[0059] An array of attachment sites on an optically transparent
solid support can be constructed using lithographic techniques
commonly used in the construction of electronic integrated circuits
as described in, e.g., techniques for attachment described in U.S.
Pat. Nos. 5,143,854, 5,445,934, 5,744,305, and 5,800,992; Chee et
al., Science 274: 610-614 (1996); Fodor et al., Nature 364: 555-556
(1993); Fodor et al., Science 251: 767-773 (1991); Gushin, et al.,
Anal. Biochem. 250: 203-211 (1997); Kinosita et al., Cell 93: 21-24
(1998); Kato-Yamada et al., J. Biol. Chem. 273: 19375-19377 (1998);
and Yasuda et al., Cell 93: 1117-1124 (1998). Photolithography and
electron beam lithography sensitize the solid support or substrate
with a linking group that allows attachment of a modified
biomolecule (e.g., proteins or nucleic acids). See e.g., Service,
Science 283: 27-28 (1999); Rai-Choudhury, HANDBOOK OF
MICROLITHOGRAPHY, MICROMACHINING, AND MICROFABRICATION, VOLUME I:
MICROLITHOGRAPHY, Volume PM39, SPIE Press (1997). Alternatively, an
array of sensitized sites can be generated using thin-film
technology as described in Zasadzinski et al., Science 263:
1726-1733 (1994).
Fiber Optic Substrate Arrays
[0060] The substrate material is preferably made of a material that
facilitates detection of the reaction event. For example, in a
typical sequencing reaction, binding of a dNTP to a sample nucleic
acid to be sequenced can be monitored by detection of photons
generated by enzyme action on phosphate liberated in the sequencing
reaction. Thus, having the substrate material made of a transparent
or optically (i.e., light) conductive material facilitates
detection of the photons.
[0061] In some embodiments, the solid support can be coupled to a
bundle of optical fibers that are used to detect and transmit the
light product. The total number of optical fibers within the bundle
may be varied so as to match the number of individual reaction
chambers in the array utilized in the sequencing reaction. The
number of optical fibers incorporated into the bundle is designed
to match the resolution of a detection device so as to allow 1:1
imaging. The overall sizes of the bundles are chosen so as to
optimize the usable area of the detection device while maintaining
desirable reagent (flow) characteristics in the reaction chamber.
Thus, for a 4096.times.4096 pixel CCD (charge-coupled device) array
with 15 .mu.m pixels the fiber bundle is chosen to be approximately
60 mm.times.60 mm or to have a diameter of approximately 90 mm. The
desired number of optical fibers are initially fused into a bundle
or optical fiber array, the terminus of which can then be cut and
polished so as to form a "wafer" of the required thickness (e.g.,
1.5 mm). The resulting optical fiber wafers possess similar
handling properties to that of a plane of glass. The individual
fibers can be any size diameter (e.g., 3 .mu.m to 100 .mu.m).
[0062] In some embodiments two fiber optic bundles are used: a
first bundle is attached directly to the detection device (also
referred to herein as the fiber bundle or connector) and a second
bundle is used as the reaction chamber substrate (the wafer or
substrate). In this case the two are placed in direct contact,
optionally with the use of optical coupling fluid, in order to
image the reaction centers onto the detection device. If a CCD is
used as the detection device, the wafer could be slightly larger in
order to maximize the use of the CCD area, or slightly smaller in
order to match the format of a typical microscope slide--25
mm.times.75 mm. The diameters of the individual fibers within the
bundles are chosen so as to maximize the probability that a single
reaction will be imaged onto a single pixel in the detection
device, within the constraints of the state of the art. Exemplary
diameters are 6-8 .mu.m for the fiber bundle and 6-50 .mu.m for the
wafer, though any diameter in the range 3-100 .mu.m can be used.
Fiber bundles can be obtained commercially from CCD camera
manufacturers. In these arrays, typically the distance between the
top surface and the bottom surface is no greater than 10 cm,
preferably no greater than 3 cm, most preferably no greater than 2
cm, and usually between 0.5 mm to 5 mm. For example, the wafer can
be obtained from Incom, Inc. (Charlton, Mass.) and cut and polished
from a large fusion of fiber optics, typically being 2 mm thick,
though possibly being 0.5 to 5 mm thick. The wafer has handling
properties similar to a pane of glass or a glass microscope
slide.
[0063] Reaction chambers can be formed in the substrate made from
fiber optic material. The surface of the optical fiber is cavitated
by treating the termini of a bundle of fibers, e.g., with acid, to
form an indentation in the fiber optic material. Thus, in one
embodiment cavities are formed from a fiber optic bundle,
preferably cavities can be formed by etching one end of the fiber
optic bundle. Each cavitated surface can form a reaction chamber.
Such arrays are referred to herein as fiber optic reactor arrays or
FORA. The indentation ranges in depth from approximately one-half
the diameter of an individual optical fiber up to two to three
times the diameter of the fiber. Cavities can be introduced into
the termini of the fibers by placing one side of the optical fiber
wafer into an acid bath for a variable amount of time. The amount
of time can vary depending upon the overall depth of the reaction
cavity desired (see e.g., Walt, et al., 1996. Anal. Chem. 70:
1888). A wide channel cavity can have uniform flow velocity
dimensions of approximately 14 mm.times.43 mm. Thus, with this
approximate dimension and at approximately 4.82.times.10.sup.-4
cavities/um.sup.2 density, the apparatus can have approximately
290,000 fluidically accessible cavities. Several methods are known
in the art for attaching molecules (and detecting the attached
molecules) in the cavities etched in the ends of fiber optic
bundles. See, e.g., Michael, et al., Anal. Chem. 70: 1242-1248
(1998); Ferguson, et al., Nature Biotechnology 14: 1681-1684
(1996); Healey and Walt, Anal. Chem. 69: 2213-2216 (1997). A
pattern of reactive sites can also be created in the microwell,
using photolithographic techniques similar to those used in the
generation of a pattern of reaction pads on a planar support. See,
Healey, et al., Science 269: 1078-1080 (1995); Munkholm and Walt,
Anal. Chem. 58: 1427-1430 (1986), and Bronk, et al., Anal. Chem.
67: 2750-2757 (1995).
[0064] The opposing side of the optical fiber wafer (i.e., the
non-etched side) is typically highly polished so as to allow
optical-coupling (e.g., by immersion oil or other optical coupling
fluids) to a second, optical fiber bundle. This second optical
fiber bundle exactly matches the diameter of the optical wafer
containing the reaction chambers, and serve to act as a conduit for
the transmission of light product to the attached detection device,
such as a CCD imaging system or camera.
[0065] In one preferred embodiment, the fiber optic wafer is
thoroughly cleaned, e.g. by serial washes in 15% H.sub.2O.sub.2/15%
NH.sub.4OH volume:volume in aqueous solution, then six deionized
water rinses, then 0.5M EDTA, then six deionized water, then 15%
H.sub.2O.sub.2/15% NH.sub.4OH, then six deionized water (one-half
hour incubations in each wash).
[0066] The surface of the fiber optic wafer is preferably coated to
facilitate its use in the sequencing reactions. A coated surface is
preferably optically transparent, allows for easy attachment of
proteins and nucleic acids, and does not negatively affect the
activity of immobilized proteins. In addition, the surface
preferably minimizes non-specific absorption of macromolecules and
increases the stability of linked macromolecules (e.g., attached
nucleic acids and proteins).
[0067] Suitable materials for coating the array include, e.g.,
plastic (e.g. polystyrene). The plastic can be preferably
spin-coated or sputtered (0.1 .mu.m thickness). Other materials for
coating the array include gold layers, e.g. 24 karat gold, 0.1
.mu.m thickness, with adsorbed self-assembling monolayers of long
chain thiol alkanes. Biotin is then coupled covalently to the
surface and saturated with a biotin-binding protein (e.g.
streptavidin or avidin).
[0068] Coating materials can additionally include those systems
used to attach an anchor primer to a substrate. Organosilane
reagents, which allow for direct covalent coupling of proteins via
amino, sulfhydryl or carboxyl groups, can also be used to coat the
array. Additional coating substances include photoreactive linkers,
e.g. photobiotin, (Amos et al., "Biomaterial Surface Modification
Using Photochemical Coupling Technology," in Encyclopedic Handbook
of Biomaterials and Bioengineering, Part A: Materials, Wise et al.
(eds.), New York, Marcel Dekker, pp. 895926, 1995).
[0069] Additional coating materials include hydrophilic polymer
gels (polyacrylamide, polysaccharides), which preferably polymerize
directly on the surface or polymer chains covalently attached post
polymerization (Hjerten, J. Chromatogr. 347, 191 (1985); Novotny,
Anal. Chem. 62, 2478 (1990), as well as pluronic polymers (triblock
copolymers, e.g. PPO-PEO-PPO, also known as F-108), specifically
adsorbed to either polystyrene or silanized glass surfaces (Ho et
al., Langmuir 14:3889-94, 1998), as well as passively adsorbed
layers of biotin-binding proteins. The surface can also be coated
with an epoxide which allows the coupling of reagents via an amine
linkage.
[0070] In addition, any of the above materials can be derivatized
with one or more functional groups, commonly known in the art for
the immobilization of enzymes and nucleotides, e.g. metal chelating
groups (e.g. nitrilo triacetic acid, iminodiacetic acid,
pentadentate chelator), which will bind 6.times.His-tagged proteins
and nucleic acids.
[0071] Surface coatings can be used that increase the number of
available binding sites for subsequent treatments, e.g. attachment
of enzymes (discussed later), beyond the theoretical binding
capacity of a 2D surface.
[0072] In a preferred embodiment, the individual optical fibers
utilized to generate the fused optical fiber bundle/wafer are
larger in diameter (i.e., 6 .mu.m to 12 .mu.m) than those utilized
in the optical imaging system (i.e., 3 .mu.m). Thus, several of the
optical imaging fibers can be utilized to image a single reaction
site.
Summary of the Arrays of This Invention
[0073] In one aspect, the invention involves an array including a
planar surface with a plurality of reaction chambers disposed
thereon, wherein the reaction chambers have a center to center
spacing of between 5 to 200 .mu.m and each chamber has a width in
at least one dimension of between 0.3 .mu.m and 100 .mu.m. In some
embodiments, the array is a planar surface with a plurality of
cavities thereon, where each cavity forms an analyte reaction
chamber. In a preferred embodiment, the array is fashioned from a
sliced fiber optic bundle (i.e., a bundle of fused fiber optic
cables) and the reaction chambers are formed by etching one surface
of the fiber optic reactor array ("FORA"). The cavities can also be
formed in the substrate via etching, molding or micromachining.
[0074] Specifically, each reaction chamber in the array typically
has a width in at least one dimension of between 0.3 .mu.m and 100
.mu.m, preferably between 0.3 .mu.m and 20 .mu.m, mst preferably
between 0.3 .mu.m and 10 .mu.m. In a separate embodiment, we
contemplate larger reaction chambers, preferably having a width in
at least one dimension of between 20 .mu.m and 70 .mu.m.
[0075] The array typically contains more than 1,000 reaction
chambers, preferably more than 400,000, more preferably between
400,000 and 20,000,000, and most preferably between 1,000,000 and
16,000,000 cavities or reaction chambers. The shape of each cavity
is frequently substantially hexagonal, but the cavities can also be
cylindrical.. In some embodiments, each cavity has a smooth wall
surface, however, we contemplate that each cavity may also have at
least one irregular wall surface. The bottom of each of the
cavities can be planar or concave.
[0076] The array is typically constructed to have cavities or
reaction chambers with a center-to-center spacing between 10 to 150
.mu.m, preferably between 50 to 100 .mu.m.
[0077] Each cavity or reaction chamber typically has a depth of
between 10 .mu.m and 100 .mu.m; alternatively, the depth is between
0.25 and 5 times the size of the width of the cavity, preferably
between 0.3 and 1 times the size of the width of the cavity.
[0078] In one embodiment, the arrays described herein typically
include a planar top surface and a planar bottom surface, which is
optically conductive such that optical signals from the reaction
chambers can be detected through the bottom planar surface. In
these arrays, typically the distance between the top surface and
the bottom surface is no greater than 10 cm, preferably no greater
than 3 cm, most preferably no greater than 2 cm.
[0079] In one embodiment, each cavity of the array contains
reagents for analyzing a nucleic acid or protein. The array can
also include a second surface spaced apart from the planar array
and in opposing contact therewith such that a flow chamber is
formed over the array.
[0080] In another aspect, the invention involves an array means for
carrying out separate parallel common reactions in an aqueous
environment, wherein the array means includes a substrate having at
least 1,000 discrete reaction chambers. These chambers contain a
starting material that is capable of reacting with a reagent. Each
of the reaction chambers are dimensioned such that when one or more
fluids containing at least one reagent is delivered into each
reaction chamber, the diffusion time for the reagent to diffuse out
of the well exceeds the time required for the starting material to
react with the reagent to form a product. The reaction chambers can
be formed by generating a plurality of cavities on the substrate,
or by generating discrete patches on a planar surface, the patches
having a different surface chemistry than the surrounding planar
surface.
[0081] In one embodiment, each cavity or reaction chamber of the
array contains reagents for analyzing a nucleic acid or protein.
Typically those reaction chambers that contain a nucleic acid (not
all reaction chambers in the array are required to) contain only a
single species of nucleic acid (i.e., a single sequence that is of
interest). There may be a single copy of this species of nucleic
acid in any particular reaction chamber, or they may be multiple
copies. It is generally preferred that a reaction chamber contain
at least 100 copies of a nucleic acid sequence, preferably at least
100,000 copies, and most preferably between 100,000 to 1,000,000
copies of the nucleic acid. The ordinarily skilled artisan will
appreciate that changes in the number of copies of a nucleic acid
species in any one reaction chamber will affect the number of
photons generated in a pyrosequencing reaction, and can be
routinely adjusted to provide more or less photon signal as is
required.
[0082] In one embodiment the nucleic acid species is amplified to
provide the desired number of copies using PCR, RCA, ligase chain
reaction, other isothermal amplification, or other conventional
means of nucleic acid amplification. In one embodimant, the nucleic
acid is single stranded. In other embodiments the single stranded
DNA is a concatamer with each copy covalently linked end to
end.
Delivery Means
[0083] An example of the means for delivering reactants to the
reaction chamber is the perfusion chamber of the present invention
is illustrated in FIG. 3. The perfusion chamber includes a sealed
compartment with transparent upper and lower slide. It is designed
to allow flow of solution over the surface of the substrate surface
and to allow for fast exchange of reagents. Thus, it is suitable
for carrying out, for example, the pyrophosphate sequencing
reactions. The shape and dimensions of the chamber can be adjusted
to optimize reagent exchange to include bulk flow exchange,
diffusive exchange, or both in either a laminar flow or a turbulent
flow regime.
[0084] The correct exchange of reactants to the reaction chamber is
important for accurate measurements in the present invention. In
the absence of convective flow of bulk fluid, transport of reaction
participants (and cross-contamination or "cross-talk" between
adjacent reaction sites or microvessels) can take place only by
diffusion. If the reaction site is considered to be a point source
on a 2-D surface, the chemical species of interest (e.g., a
reaction product) will diffuse radially from the site of its
production, creating a substantially hemispherical concentration
field above the surface.
[0085] The distance that a chemical entity can diffuse in any given
time t may be estimated in a crude manner by considering the
mathematics of diffusion (Crank, The Mathematics of Diffusion,
2.sup.nd ed. 1975). The rate of diffusive transport in any given
direction x (cm) is given by Fick's law as j = - D .times.
.differential. C .differential. x Eq . .times. 1 ##EQU1## where j
is the flux per unit area (g-mol/cm.sup.2-s) of a species with
diffusion coefficient D (cm.sup.2/s), and
.differential.c/.differential.x is the concentration gradient of
that species. The mathematics of diffusion are such that a
characteristic or "average" distance an entity can travel by
diffusion alone scales with the one-half power of both the
diffusion coefficient and the time allowed for diffusion to occur.
Indeed, to order of magnitude, this characteristic diffusion
distance can be estimated as the square root of the product of the
diffusion coefficient and time--as adjusted by a numerical factor
of order unity that takes into account the particulars of the
system geometry and initial and/or boundary conditions imposed on
the diffusion process.
[0086] It will be convenient to estimate this characteristic
diffusion distance as the root-mean-square distance d.sub.rms that
a diffusing entity can travel in time t: d rms = 2 .times. Dt Eq .
.times. 2 ##EQU2##
[0087] As stated above, the distance that a diffusing chemical
typically travels varies with the square root of the time available
for it to diffuse--and inversely, the time required for a diffusing
chemical to travel a given distance scales with the square of the
distance to be traversed by diffusion. Thus, for a simple,
low-molecular-weight biomolecule characterized by a diffusion
coefficient D of order 110.sup.-5 cm.sup.2/s, the root-mean-square
diffusion distances d.sub.rms that can be traversed in time
intervals of 0.1 s, 1.0 s, 2.0 s, and 10 s are estimated by means
of Equation 2 as 14 .mu.m, 45 .mu.m, 63 .mu.m, and 141 .mu.m,
respectively.
[0088] The relative importance of convection and diffusion in a
transport process that involves both mechanisms occurring
simultaneously can be gauged with the aid of a dimensionless
number--namely, the Peclet number Pe. This Peclet number can be
viewed as a ratio of two rates or velocities--namely, the rate of a
convective flow divided by the rate of a diffusive "flow" or flux.
More particularly, the Peclet number is a ratio of a characteristic
flow velocity V(in cm/s) divided by a characteristic diffusion
velocity D/L (also expressed in units of cm/s)--both taken in the
same direction: Pe = VL D Eq . .times. 3 ##EQU3##
[0089] In Equation 3, V is the average or characteristic speed of
the convective flow, generally determined by dividing the
volumetric flow rate Q (in cm.sup.3/s) by the cross-sectional area
A (cm.sup.2) available for flow. The characteristic length L is a
representative distance or system dimension measured in a direction
parallel to the directions of flow and of diffusion (i.e., in the
direction of the steepest concentration gradient) and selected to
be representative of the typical or "average" distance over which
diffusion occurs in the process. And finally D (cm.sup.2/s) is the
diffusion coefficient for the diffusing species in question. (An
alternative but equivalent formulation of the Peclet number Pe
views it as the ratio of two characteristic times--namely, of
representative times for diffusion and convection. Equation 3 for
the Peclet number can equally well be obtained by dividing the
characteristic diffusion time L.sup.2/D by the characteristic
convection time L/V.)
[0090] The convective component of transport can be expected to
dominate over the diffusive component in situations where the
Peclet number Pe is large compared to unity. Conversely, the
diffusive component of transport can be expected to dominate over
the convective component in situations where the Peclet number Pe
is small compared to unity. In extreme situations where the Peclet
number is either very much larger or very much smaller than one,
transport may be accurately presumed to occur either by convection
or by diffusion alone, respectively. Finally, in situations where
the estimated Peclet number is of order unity, then both convection
and diffusion can be expected to play significant roles in the
overall transport process.
[0091] The diffusion coefficient of a typical low-molecular-weight
biomolecule will generally be of the order of 10.sup.-5 cm.sup.2/s
(e.g., 0.5210.sup.-5 Cm/s for sucrose, and 1.0610.sup.-5 cm/s for
glycine). Thus, for reaction centers, cavities, or wells separated
by a distance of 100 .mu.m (i.e.. 0.01 cm), the Peclet number Pe
for low-molecular-weight solutes such as these will exceed unity
for flow velocities greater than about 10 .mu.m/sec (0.001 cm/s).
For cavities separated by only 10 .mu.m (i.e., 0.001 cm), the
Peclet number Pe for low-molecular-weight solutes will exceed unity
for flow velocities greater than about 100 .mu.m/sec (0.01 cm/s).
Convective transport is thus seen to dominate over diffusive
transport for all but very slow flow rates and/or very short
diffusion distances.
[0092] Where the molecular weight of a diffusible species is
substantially larger--for example as it is with large biomolecules
like DNA/RNA, DNA fragments, oligonucleotides, proteins, and
constructs of the former--then the species diffusivity will be
corresponding smaller, and convection will play an even more
important role relative to diffusion in a transport process
involving both mechanisms. For instance, the aqueous-phase
diffusion coefficients of proteins fall in about a 10-fold range
(Tanford, Physical Chemistry of Macromolecules, 1961). Protein
diffusivities are bracketed by values of 1.19.times.10.sup.-6
cm.sup.2/s for ribonuclease (a small protein with a molecular
weight of 13,683 Daltons) and 1.16.times.10.sup.-7 cm.sup.2/s for
myosin (a large protein with a molecular weight of 493,000
Daltons). Still larger entities (e.g., tobacco mosaic virus or TMV
at 40.6 million Daltons) are characterized by still lower
diffusivities (in particular, 4.6.times.10.sup.-8 cm.sup.2/s for
TMV) (Lehninger, Biochemistry, 2.sup.nd ed. 1975). The fluid
velocity at which convection and diffusion contribute roughly
equally to transport (i.e., Pe of order unity) scales in direct
proportion to species diffusivity.
[0093] With the aid of the Peclet number formalism it is possible
to gauge the impact of convection on reactant supply to--and
product removal from--reaction chambers, cavities or wells. On the
one hand, it is clear that even modest convective flows can
appreciably increase the speed at which reactants are delivered to
the interior of the cavities in an array or FORA. In particular,
suppose for the sake of simplicity that the criteria for roughly
equal convective and diffusive flows is considered to be Pe=1. One
may then estimate that a convective flow velocity of the order of
only 0.004 cm/s will suffice to carry reactant into a 25-.mu.m-deep
well at roughly the same rate as it could be supplied to the bottom
of the well by diffusion alone, given an assumed value for reactant
diffusivity of 1.times.10.sup.-5 cm.sup.2/s. The corresponding flow
velocity required to match the rate of diffusion of such a species
from the bottom to the top of a 2.5-.mu.m-deep microwell is
estimated to be of order 0.04 cm/s. Flow velocities through a FORA
much higher than this are possible, thereby illustrating the degree
to which a modest convective flow can augment the diffusive supply
of reactants to FORA reaction centers, cavities or wells.
[0094] The perfusion chamber is preferably detached from the
imaging system while it is being prepared and only placed on the
imaging system when sequencing analysis is performed. In one
embodiment, the solid support (i.e., a DNA chip or glass slide) is
held in place by a metal or plastic housing, which may be assembled
and disassembled to allow replacement of said solid support. The
lower side of the solid support of the perfusion chamber carries
the reaction chamber array and, with a traditional optical-based
focal system, a high numerical aperture objective lens is used to
focus the image of the reaction center array onto the CCD imaging
system.
[0095] An alternative system for the analysis is to use an array
format wherein samples are distributed over a surface, for example
a microfabricated chip, and thereby an ordered set of samples may
be immobilized in a 2-dimensional format. Many samples can thereby
be analyzed in parallel. Using the method of the invention, many
immobilized templates may be analyzed in this was by allowing the
solution containing the enzymes and one nucleotide to flow over the
surface and then detecting the signal produced for each sample.
This procedure can then be repeated. Alternatively, several
different oligonucleotides complementary to the template may be
distributed over the surface followed by hybridization of the
template. Incorporation of deoxynucleotides or dideoxynucleotides
may be monitored for each oligonucleotide by the signal produced
using the various oligonucleotides as primer. By combining the
signals from different areas of the surface, sequence-based
analyses may be performed by four cycles of polymerase reactions
using the various dideoxynucleotides.
[0096] When the support is in the form of a cavitated array, e.g.,
in the termini of a FORA or other array of microwells, suitable
delivery means for reagents include flowing and washing and also,
e.g., flowing, spraying, electrospraying, ink jet delivery,
stamping, ultrasonic atomization (Sonotek Corp., Milton, N.Y.) and
rolling. Preferably, all reagent solutions contain 10-20% ethylene
glycol to minimize evaporation. When spraying is used, reagents are
delivered to the FORA surface in a homogeneous thin layer produced
by industrial type spraying nozzles (Spraying Systems. Co.,
Wheaton, Ill.) or atomizers used in thin layer chromatography
(TLC), such as CAMAG TLC Sprayer (Camag Scientific Inc.,
Wilmington, N.C.). These sprayers atomize reagents into aerosol
spray particles in the size range of 0.3 to 10 .mu.m.
[0097] Electrospray deposition (ESD) of protein and DNA solutions
is currently used to generate ions for mass spectrometric analysis
of these molecules. Deposition of charged electrospray products on
certain areas of a FORA substrate under control of electrostatic
forces is suggested. It was also demonstrated that the ES-deposited
proteins and DNA retain their ability to specifically bind
antibodies and matching DNA probes, respectively, enabling use of
the ESD fabricated matrixes in Dot Immuno-Binding (DIB) and in DNA
hybridization assays. (Morozov and Morozova Anal. Chem.
71(15):3110-7 (1999)).
[0098] Ink-jet delivery is applicable to protein solutions and
other biomacromolecules, as documented in the literature (e.g. Roda
et al., Biotechniques 28(3): 492-6 (2000)). It is also commercially
available e.g. from MicroFab Technologies, Inc. (Plano, Tex.).
[0099] Reagent solutions can alternatively be delivered to the FORA
surface by a method similar to lithography. Rollers (stamps;
hydrophilic materials should be used) would be first covered with a
reagent layer in reservoirs with dampening sponges and then rolled
over (pressed against) the FORA surface.
[0100] Successive reagent delivery steps are preferably separated
by wash steps using techniques commonly known in the art. These
washes can be performed, e.g., using the above described methods,
including high-flow sprayers or by a liquid flow over the FORA or
microwell array surface. The washes can occur in any time period
after the starting material has reacted with the reagent to form a
product in each reaction chamber but before the reagent delivered
to any one reaction chamber has diffused out of that reaction
chamber into any other reaction chamber. In one embodiment, any one
reaction chamber is independent of the product formed in any other
reaction chamber, but is generated using one or more common
reagents.
[0101] An embodiment of a complete apparatus is illustrated in FIG.
2. The apparatus includes an inlet conduit 200 in communication
with a detachable perfusion chamber 226. The inlet conduit 200
allows for entry of sequencing reagents via a plurality of tubes
202-212, which are each in communication with a plurality of
sequencing dispensing reagent vessels 214-224.
[0102] Reagents are introduced through the conduit 200 into the
perfusion chamber 226 using either a pressurized system or pumps to
drive positive flow. Typically, the reagent flow rates are from
0.05 to 50 ml/minute (e.g., 1 to 50 ml/minute) with volumes from
0.100 ml to continuous flow (for washing). Valves are under
computer control to allow cycling of nucleotides and wash reagents.
Sequencing reagents, e.g., polymerase can be either pre-mixed with
nucleotides or added in stream. A manifold brings all six tubes
202-212 together into one for feeding the perfusion chamber. Thus
several reagent delivery ports allow access to the perfusion
chamber. For example, one of the ports may be utilized to allow the
input of the aqueous sequencing reagents, while another port allows
these reagents (and any reaction products) to be withdrawn from the
perfusion chamber.
[0103] The perfusion chamber 226 contains the substrate comprising
the plurality of reaction chambers. The perfusion chamber allows
for a uniform, linear flow of the required sequencing reagents, in
aqueous solution, over the amplified nucleic acids and allows for
the rapid and complete exchange of these reagents. Thus, it is
suitable for performing pyrophosphate-based sequencing reactions.
The perfusion chamber can also be used to prepare the anchor
primers and perform amplification reactions, e.g., the RCA
reactions described herein.
[0104] The invention also provides a method for delivering nucleic
acid sequencing enzymes to an array. In some embodiments, one of
the nucleic acid sequencing enzymes can be a polypeptide with
sulfurylase activity or the nucleic acid sequencing enzyme can be a
polypeptide with luciferase activity. In another embodiment, one of
the nucleic acid sequencing enzymes can be a polypeptide with both
sulfurylase and luciferase activity. In a more preferred
embodiment, the reagent can be suitable for use in a nucleic acid
sequencing reaction.
[0105] In a preferred embodiment, one or more reagents are
delivered to an array immobilized or attached to a population of
mobile solid supports, e.g., a bead or microsphere. The bead or
microsphere need not be spherical, irregular shaped beads may be
used. They are typically constructed from numerous substances,
e.g., plastic, glass or ceramic and bead sizes ranging from
nanometers to millimeters depending on the width of the reaction
chamber. Preferably, the diameter of each mobile solid support can
be between 0.01 and 0.1 times the width of each cavity. Various
bead chemistries can be used e.g., methylstyrene, polystyrene,
acrylic polymer, latex, paramagnetic, thoria sol, carbon graphite
and titanium dioxide. The construction or chemistry of the bead can
be chosen to facilitate the attachment of the desired reagent.
[0106] In another embodiment, the bioactive agents are synthesized
first, and then covalently attached to the beads. As is appreciated
by someone skilled in the art, this will be done depending on the
composition of the bioactive agents and the beads. The
functionalization of solid support surfaces such as certain
polymers with chemically reactive groups such as thiols, amines,
carboxyls, etc. is generally known in the art. Accordingly, "blank"
beads may be used that have surface chemistries that facilitate the
attachment of the desired functionality by the user. Additional
examples of these surface chemistries for blank beads include, but
are not limited to, amino groups including aliphatic and aromatic
amines, carboxylic acids, aldehydes, amides, chloromethyl groups,
hydrazide, hydroxyl groups, sulfonates and sulfates.
[0107] These functional groups can be used to add any number of
different candidate agents to the beads, generally using known
chemistries. For example, candidate agents containing carbohydrates
may be attached to an amino-functionalized support; the aldehyde of
the carbohydrate is made using standard techniques, and then the
aldehyde is reacted with an amino group on the surface. In an
alternative embodiment, a sulfhydryl linker may be used. There are
a number of sulfhydryl reactive linkers known in the art such as
SPDP, maleimides, .alpha.-haloacetyls, and pyridyl disulfides (see
for example the 1994 Pierce Chemical Company catalog, technical
section on cross-linkers, pages 155-200, incorporated here by
reference) which can be used to attach cysteine containing
proteinaceous agents to the support. Alternatively, an amino group
on the candidate agent may be used for attachment to an amino group
on the surface. For example, a large number of stable bifunctional
groups are well known in the art, including homobifunctional and
heterobifunctional linkers (see Pierce Catalog and Handbook, pages
155-200). In an additional embodiment, carboxyl groups (either from
the surface or from the candidate agent) may be derivatized using
well known linkers (see Pierce catalog). For example, carbodiimides
activate carboxyl groups for attack by good nucleophiles such as
amines (see Torchilin et al., Critical Rev. Thereapeutic Drug
Carrier Systems, 7(4):275-308 (1991)). Proteinaceous candidate
agents may also be attached using other techniques known in the
art, for example for the attachment of antibodies to polymers; see
Slinkin et al., Bioconj. Chem. 2:342-348 (1991); Torchilin et al.,
supra; Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et
al., Cancer Res. 54:6176-6185 (1994); and Wilbur et al.,
Bioconjugate Chem. 5:220-235 (1994). It should be understood that
the candidate agents may be attached in a variety of ways,
including those listed above. Preferably, the manner of attachment
does not significantly alter the functionality of the candidate
agent; that is, the candidate agent should be attached in such a
flexible manner as to allow its interaction with a target.
[0108] Specific techniques for immobilizing enzymes on beads are
known in the prior art. In one case, NH.sub.2 surface chemistry
beads are used. Surface activation is achieved with a 2.5%
glutaraldehyde in phosphate buffered saline (10 mM) providing a pH
of 6-9 (138 mM NaCl, 2.7 mM KCl). This mixture is stirred on a stir
bed for approximately 2 hours at room temperature. The beads are
then rinsed with ultrapure water plus 0.01% Tween 20 (surfactant)
-0.02%, and rinsed again with a pH 7.7 PBS plus 0.01% tween 20.
Finally, the enzyme is added to the solution, preferably after
being prefiltered using a 0.45 .mu.m amicon micropure filter.
[0109] The population of mobile solid supports are disposed in the
reaction chambers. In some embodiments, 5% to 20% of the reaction
chambers can have a mobile solid support with at least one reagent
immobilized thereon, 20% to 60% of the reaction chambers can have a
mobile solid support with at least one reagent immobilized thereon
or 50% to 100% of the reaction chambers can have a mobile solid
support with at least one reagent immobilized thereon. Preferably,
at least one reaction chamber has a mobile solid support having at
least one reagent immobilized thereon and the reagent is suitable
for use in a nucleic acid sequencing reaction.
[0110] In some embodiments, the reagent immobilized to the mobile
solid support can be a polypeptide with sulfurylase activity, a
polypeptide with luciferase activity or a chimeric polypeptide
having both sulfurylase and luciferase activity. In one embodiment,
it can be a ATP sulfurylase and luciferase fusion protein. Since
the product of the sulfurylase reaction is consumed by luciferase,
proximity between these two enzymes may be achieved by covalently
linking the two enzymes in the form of a fusion protein. This
invention would be useful not only in substrate channeling but also
in reducing production costs and potentially doubling the number of
binding sites on streptavidin-coated beads.
[0111] In another embodiment, the sulfurylase is a thermostable ATP
sulfurylase. In a preferred embodiment, the thermostable
sulfurylase is active at temperatures above ambient (to at least
50.degree. C.). In one embodiment, the ATP sulfurylase is from a
thermophile. In an additional embodiment, the mobile solid support
can have a first reagent and a second reagent immobilized thereon,
the first reagent is a polypeptide with sulfurylase activity and
the second reagent is a polypeptide with luciferase activity.
[0112] In another embodiment, the reagent immobilized to the mobile
solid support can be a nucleic acid; preferably the nucleic acid is
a single stranded concatamer. In a preferred embodiment, the
nucleic acid can be used for sequencing a nucleic acid, e.g., a
pyrosequencing reaction.
[0113] The invention also provides a method for detecting or
quantifying ATP activity using a mobile solid support; preferably
the ATP can be detected or quantified as part of a nucleic acid
sequencing reaction.
[0114] A FORA that has been "carpeted" with mobile solid supports
with either nucleic acid or reagent enzymes attached thereto is
shown as FIG. 7.
[0115] The solid support is optically linked to an imaging system
230, which includes a CCD system in association with conventional
optics or a fiber optic bundle. In one embodiment the perfusion
chamber substrate includes a fiber optic array wafer such that
light generated near the aqueous interface is transmitted directly
through the optical fibers to the exterior of the substrate or
chamber. When the CCD system includes a fiber optic connector,
imaging can be accomplished by placing the perfusion chamber
substrate in direct contact with the connector. Alternatively,
conventional optics can be used to image the light, e.g., by using
a 1-1 magnification high numerical aperture lens system, from the
exterior of the fiber optic substrate directly onto the CCD sensor.
When the substrate does not provide for fiber optic coupling, a
lens system can also be used as described above, in which case
either the substrate or the perfusion chamber cover is optically
transparent. An exemplary CCD imaging system is described
above.
[0116] The imaging system 230 is used to collect light from the
reactors on the substrate surface. Light can be imaged, for
example, onto a CCD using a high sensitivity low noise apparatus
known in the art. For fiber-optic based imaging, it is preferable
to incorporate the optical fibers directly into the cover slip or
for a FORA to have the optical fibers that form the microwells also
be the optical fibers that convey light to the detector.
[0117] The imaging system is linked to a computer control and data
collection system 240. In general, any commonly available hardware
and software package can be used. The computer control and data
collection system is also linked to the conduit 200 to control
reagent delivery.
[0118] The photons generated by the pyrophosphate sequencing
reaction are captured by the CCD only if they pass through a
focusing device (e.g., an optical lens or optical fiber) and are
focused upon a CCD element. However, the emitted photons will
escape equally in all directions. In order to maximize their
subsequent "capture" and quantitation when utilizing a planar array
(e.g., a DNA chip), it is preferable to collect the photons as
close as possible to the point at which they are generated, e.g.
immediately at the planar solid support. This is accomplished by
either: (i) utilizing optical immersion oil between the cover slip
and a traditional optical lens or optical fiber bundle or,
preferably, (ii) incorporating optical fibers directly into the
cover slip itself. Similarly, when a thin, optically transparent
planar surface is used, the optical fiber bundle can also be placed
against its back surface, eliminating the need to "image" through
the depth of the entire reaction/perfusion chamber.
Detection Means
[0119] The reaction event, e.g., photons generated by luciferase,
may be detected and quantified using a variety of detection
apparatuses, e.g., a photomultiplier tube, a CCD, CMOS, absorbance
photometer, a luminometer, charge injection device (CID), or other
solid state detector, as well as the apparatuses described herein.
In a preferred embodiment, the quantitation of the emitted photons
is accomplished by the use of a CCD camera fitted with a fused
fiber optic bundle. In another preferred embodiment, the
quantitation of the emitted photons is accomplished by the use of a
CCD camera fitted with a microchannel plate intensifier. A
back-thinned CCD can be used to increase sensitivity. CCD detectors
are described in, e.g., Bronks, et al., 1995. Anal. Chem. 65:
2750-2757.
[0120] An exemplary CCD system is a Spectral Instruments, Inc.
(Tucson, Ariz.) Series 600 4-port camera with a Lockheed-Martin
LM485 CCD chip and a 1-1 fiber optic connector (bundle) with 6-8
.mu.m individual fiber diameters. This system has 4096.times.4096,
or greater than 16 million pixels and has a quantum efficiency
ranging from 10% to >40%. Thus, depending on wavelength, as much
as 40% of the photons imaged onto the CCD sensor are converted to
detectable electrons.
[0121] In other embodiments, a fluorescent moiety can be used as a
label and the detection of a reaction event can be carried out
using a confocal scanning microscope to scan the surface of an
array with a laser or other techniques such as scanning near-field
optical microscopy (SNOM) are available which are capable of
smaller optical resolution, thereby allowing the use of "more
dense" arrays. For example, using SNOM, individual polynucleotides
may be distinguished when separated by a distance of less than 100
nm, e.g., 10 nm.times.10 nm. Additionally, scanning tunneling
microscopy (Binning et al., Helvetica Physica Acta, 55:726-735,
1982) and atomic force microscopy (Hanswa et al., Annu Rev Biophys
Biomol Struct, 23:115-139, 1994) can be used.
[0122] The invention provides an apparatus for simultaneously
monitoring an array of reaction chambers for light indicating that
a reaction is taking place at a particular site. The apparatus can
include an array of reaction chambers formed from a planar
substrate having a plurality of cavitated surfaces, each cavitated
surface forming a reaction chamber adapted to contain analytes. The
reaction chambers can have a center-to-center spacing of between 5
to 200 .mu.m and the array can have more than 400,000 discrete
reaction chambers. The apparatus can also include an optically
sensitive device arranged so that in use the light from a
particular reaction chamber will impinge upon a particular
predetermined region of said optically sensitive device. The
apparatus can further include a means for determining the light
level impinging upon each predetermined region and a means to
record the variation of said light level with time for each of said
reaction chamber.
[0123] The invention also provides an analytic sensor, which can
include an array formed from a first bundle of optical fibers with
a plurality of cavitated surfaces at one end thereof, each
cavitated surface forming a reaction chamber adapted to contain
analytes. The reaction chambers can have a center-to-center spacing
of between 5 to 200 .mu.m and the array can have more than 400,000
discrete reaction chambers. The analytic sensor can also include an
enzymatic or fluorescent means for generating light in the reaction
chambers. The analytic sensor can further include a light detection
means comprising a light capture means and a second fiber optic
bundle for transmitting light to the light detecting means. The
second fiber optic bundle can be in optical contact with the array,
such that light generated in an individual reaction chamber is
captured by a separate fiber or groups of separate fibers of the
second fiber optic bundle for transmission to the light capture
means. The light capture means can be a CCD camera as described
herein. The reaction chambers can contain one or more mobile solid
supports with a bioactive agent immobilized thereon. In some
embodiments, the analytic sensor is suitable for use in a
biochemical assay or suitable for use in a cell-based assay.
Methods of Sequencing Nucleic Acids
[0124] The invention also provides a method for sequencing nucleic
acids which generally comprises (a) providing one or more nucleic
acid anchor primers and a plurality of single-stranded circular
nucleic acid templates disposed within a plurality of reaction
chambers or cavities; (b) annealing an effective amount of the
nucleic acid anchor primer to at least one of the single-stranded
circular templates to yield a primed anchor primer-circular
template complex; (c) combining the primed anchor primer-circular
template complex with a polymerase to form an extended anchor
primer covalently linked to multiple copies of a nucleic acid
complementary to the circular nucleic acid template; (d) annealing
an effective amount of a sequencing primer to one or more copies of
said covalently linked complementary nucleic acid; (e) extending
the sequencing primer with a polymerase and a predetermined
nucleotide triphosphate to yield a sequencing product and, if the
predetermined nucleotide triphosphate is incorporated onto the 3'
end of said sequencing primer, a sequencing reaction byproduct; and
(f) identifying the sequencing reaction byproduct, thereby
determining the sequence of the nucleic acid. In one embodiment,
the sequencing byproduct is PPi. In another embodiment, a dATP or
ddATP analogue is used in place of deoxy- or dideoxy adenosine
triphosphate. This analogue is capable of acting as a substrate for
a polymerase but incapable of acting as a substrate for a
PPi-detection enzyme. This method can be carried out in separate
parallel common reactions in an aqueous environment.
[0125] In another aspect, the invention includes a method of
determining the base sequence of a plurality of nucleotides on an
array, which generally comprises (a) providing a plurality of
sample DNAs, each disposed within a plurality of cavities on a
planar surface; (b) adding an activated nucleotide 5'-triphosphate
precursor of one known nitrogenous base to a reaction mixture in
each reaction chamber, each reaction mixture comprising a
template-directed nucleotide polymerase and a single-stranded
polynucleotide template hybridized to a complementary
oligonucleotide primer strand at least one nucleotide residue
shorter than the templates to form at least one unpaired nucleotide
residue in each template at the 3'-end of the primer strand, under
reaction conditions which allow incorporation of the activated
nucleoside 5'-triphosphate precursor onto the 3'-end of the primer
strands, provided the nitrogenous base of the activated nucleoside
5'-triphosphate precursor is complementary to the nitrogenous base
of the unpaired nucleotide residue of the templates; (c) detecting
whether or not the nucleoside 5'-triphosphate precursor was
incorporated into the primer strands in which incorporation of the
nucleoside 5'-triphosphate precursor indicates that the unpaired
nucleotide residue of the template has a nitrogenous base
composition that is complementary to that of the incorporated
nucleoside 5'-triphosphate precursor; and (d) sequentially
repeating steps (b) and (c), wherein each sequential repetition
adds and, detects the incorporation of one type of activated
nucleoside 5'-triphosphate precursor of known nitrogenous base
composition; and (e) determining the base sequence of the unpaired
nucleotide residues of the template in each reaction chamber from
the sequence of incorporation of said nucleoside precursors.
[0126] In one embodiment of the invention, the anchor primer is
linked to a particle. The anchor primer could be linked to the
particle prior to or after formation of the extended anchor primer.
The sequencing reaction byproduct could be PPi and a coupled
sulfurylase/luciferase reaction is used to generate light for
detection. Either or both of the sulfurylase and luciferase could
be immobilized on one or more mobile solid supports disposed at
each reaction site.
[0127] In another aspect, the invention involves, a method of
determining the base sequence of a plurality of nucleotides on an
array. The method includes providing a plurality of sample DNAs,
each disposed within a plurality of cavities on a planar surface,
each cavity forming an analyte reaction chamber, wherein the
reaction chambers have a center to center spacing of between 5 to
200 .mu.m. Then an activated nucleotide 5'-triphosphate precursor
of one known nitrogenous base is added to a reaction mixture in
each reaction chamber. Each reaction mixture includes a
template-directed nucleotide polymerase and a single-stranded
polynucleotide template hybridized to a complementary
oligonucleotide primer strand at least one nucleotide residue
shorter than the templates to form at least one unpaired nucleotide
residue in each template at the 3'-end of the primer strand, under
reaction conditions which allow incorporation of the activated
nucleoside 5'-triphosphate precursor onto the 3'-end of the primer
strands, provided the nitrogenous base of the activated nucleoside
5'-triphosphate precursor is complementary to the nitrogenous base
of the unpaired nucleotide residue of the templates. Then it is
detected whether or not the nucleoside 5'-triphosphate precursor
was incorporated into the primer strands in which incorporation of
the nucleoside 5'-triphosphate precursor indicates that the
unpaired nucleotide residue of the template has a nitrogenous base
composition that is complementary to that of the incorporated
nucleoside 5'-triphosphate precursor. Then these steps are
sequentially repeated, wherein each sequential repetition adds and,
detects the incorporation of one type of activated nucleoside
5'-triphosphate precursor of known nitrogenous base composition.
The base sequence of the unpaired nucleotide residues of the
template in each reaction chamber is then determined from the
sequence of incorporation of the nucleoside precursors.
[0128] In another aspect, the invention involves a method for
determining the nucleic acid sequence in a template nucleic acid
polymer. The method includes introducing a plurality of template
nucleic acid polymers into a plurality of cavities on a planar
surface, each cavity forming an analyte reaction chamber, wherein
the reaction chambers have a center to center spacing of between 5
to 200 .mu.m. Each reaction chamber also has a polymerization
environment in which the nucleic acid polymer will act as a
template polymer for the synthesis of a complementary nucleic acid
polymer when nucleotides are added. A series of feedstocks is
successively provided to the polymerization environment, each
feedstock having a nucleotide selected from among the nucleotides
from which the complementary nucleic acid polymer will be formed,
such that if the nucleotide in the feedstock is complementary to
the next nucleotide in the template polymer to be sequenced the
nucleotide will be incorporated into the complementary polymer and
inorganic pyrophosphate will be released. Then the formation of
inorganic pyrophosphate is detected to determine the identity of
each nucleotide in the complementary polymer and thus the sequence
of the template polymer.
[0129] In another aspect, the invention involves, a method of
identifying the base in a target position in a DNA sequence of
sample DNA. The method includes providing a sample of DNA disposed
within a plurality of cavities on a planar surface, each cavity
forming an analyte reaction chamber, wherein the reaction chambers
have a center to center spacing of between 5 to 200 .mu.m, the DNA
being rendered single stranded either before or after being
disposed in the reaction chambers. An extension primer is then
provided which hybridizes to the immobilized single-stranded DNA at
a position immediately adjacent to the target position. The
immobilized single-stranded DNA is subjected to a polymerase
reaction in the presence of a predetermined nucleotide
triphosphate, wherein if the predetermined nucleotide triphosphate
is incorporated onto the 3' end of the sequencing primer then a
sequencing reaction byproduct is formed. The sequencing reaction
byproduct is then identified, thereby determining the nucleotide
complementary to the base at the target position.
[0130] In another aspect, the invention involves a method of
identifying a base at a target position in a sample DNA sequence.
The method includes providing sample DNA disposed within a
plurality of cavities on a planar surface, each cavity forming an
analyte reaction chamber, wherein the reaction chambers have a
center to center spacing of between 5 to 200 .mu.m, the DNA being
rendered single stranded either before or after being disposed in
the reaction chambers and providing an extension primer which
hybridizes to the sample DNA immediately adjacent to the target
position. The sample DNA sequence and the extension primer are then
subjected to a polymerase reaction in the presence of a nucleotide
triphosphate whereby the nucleotide triphosphate will only become
incorporated and release pyrophosphate (PPi) if it is complementary
to the base in the target position, the nucleotide triphosphate
being added either to separate aliquots of sample-primer mixture or
successively to the same sample-primer mixture. The release of PPi
is then detected to indicate which nucleotide is incorporated.
[0131] In another aspect, the invention involves a method of
identifying a base at a target position in a single-stranded sample
DNA sequence. The method includes providing an extension primer
which hybridizes to sample DNA immediately adjacent to the target
position, the sample DNA disposed within a plurality of cavities on
a planar surface, each cavity forming an analyte reaction chamber,
wherein the reaction chambers have a center to center spacing of
between 5 to 200 um, the DNA being rendered single stranded either
before or after being disposed in the reaction chambers. The sample
DNA and extension primer is subjected to a polymerase reaction in
the presence of a predetermined deoxynucleotide or
dideoxynucleotide whereby the deoxynucleotide or dideoxynucleotide
will only become incorporated and release pyrophosphate (PPi) if it
is complementary to the base in the target position, the
predetermined deoxynucleotides or dideoxynucleotides being added
either to separate aliquots of sample-primer mixture or
successively to the same sample-primer mixture. Any release of PPi
is detected enzymatically to indicate which deoxynucleotide or
dideoxynucleotide is incorporated. Characterized in that, the
PPi-detection enzyme(s) are included in the polymerase reaction
step and in that in place of deoxy- or dideoxy adenosine
triphosphate (ATP) a dATP or ddATP analogue is used which is
capable of acting as a substrate for a polymerase but incapable of
acting as a substrate for a the PPi-detection enzyme.
[0132] In another aspect, the invention involves a method for
sequencing a nucleic acid. The method includes providing one or
more nucleic acid anchor primers; and a plurality of nucleic acid
templates disposed within a plurality of cavities on the above
described arrays. An effective amount of the nucleic acid anchor
primer is annealed to at least one of the single-stranded circular
templates to yield a primed anchor primer-circular template
complex. The primed anchor primer-circular template complex is then
combined with a polymerase to form an extended anchor primer
covalently linked to multiple copies of a nucleic acid
complementary to the circular nucleic acid template; followed by
annealing of an effective amount of a sequencing primer to one or
more copies of the covalently linked complementary nucleic acid.
The sequencing primer is then extended with a polymerase and a
predetermined nucleotide triphosphate to yield a sequencing product
and, if the predetermined nucleotide triphosphate is incorporated
onto the 3' end of the sequencing primer, a sequencing reaction
byproduct. Then the sequencing reaction byproduct is identified,
thereby determining the sequence of the nucleic acid.
Structure of Anchor Primers
[0133] The anchor primers of the invention generally comprise a
stalk region and at least one adaptor region. In a preferred
embodiment the anchor primer contains at least two contiguous
adapter regions. The stalk region is present at the 5' end of the
anchor primer and includes a region of nucleotides for attaching
the anchor primer to the solid substrate.
[0134] The adaptor region(s) comprise nucleotide sequences that
hybridize to a complementary sequence present in one or more
members of a population of nucleic acid sequences. In some
embodiments, the anchor primer includes two adjoining adaptor
regions, which hybridize to complementary regions ligated to
separate ends of a target nucleic acid sequence. This embodiment is
illustrated in FIG. 1, which is discussed in more detail below. In
additional embodiments, the adapter regions in the anchor primers
are complementary to non-contiguous regions of sequence present in
a second nucleic acid sequence. Each adapter region, for example,
can be homologous to each terminus of a fragment produced by
digestion with one or more restriction endonucleases. The fragment
can include, e.g., a sequence known or suspected to contain a
sequence polymorphism. Additionally, the anchor primer may contain
two adapter regions that are homologous to a gapped region of a
target nucleic acid sequence, i.e., one that is non-contiguous
because of a deletion of one or more nucleotides. When adapter
regions having these sequences are used, an aligning
oligonucleotide corresponding to the gapped sequence may be
annealed to the anchor primer along with a population of template
nucleic acid molecules.
[0135] The anchor primer may optionally contain additional elements
such as one or more restriction enzyme recognition sites, RNA
polymerase binding sites, e.g., a T7 promoter site, or sequences
present in identified DNA sequences, e.g., sequences present in
known genes. The adapter region(s) may also include sequences known
to flank sequence polymorphisms. Sequence polymorphisms include
nucleotide substitutions, insertions, deletions, or other
rearrangements which result in a sequence difference between two
otherwise identical nucleic acid sequences. An example of a
sequence polymorphism is a single nucleotide polymorphism
(SNP).
[0136] In general, any nucleic acid capable of base-pairing can be
used as an anchor primer. In some embodiments, the anchor primer is
an oligonucleotide. As utilized herein the term oligonucleotide
includes linear oligomers of natural or modified monomers or
linkages, e.g., deoxyribonucleosides, ribonucleosides, anomeric
forms thereof, peptide nucleic acids (PNAs), and the like, that are
capable of specifically binding to a target polynucleotide by way
of a regular pattern of monomer-to-monomer interactions. These
types of interactions can include, e.g., Watson-Crick type of
base-pairing, base stacking, Hoogsteen or reverse-Hoogsteen types
of base-pairing, or the like. Generally, the monomers are linked by
phosphodiester bonds, or analogs thereof, to form oligonucleotides
ranging in size from, e.g., 3-200, 8-150, 10-100, 20-80, or 25-50
monomeric units. Whenever an oligonucleotide is represented by a
sequence of letters, it is understood that the nucleotides are
oriented in the 5'.fwdarw.3' direction, from left-to-right, and
that the letter "A" donates deoxyadenosine, the letter "T" denotes
thymidine, the letter "C" denotes deoxycytosine, and the letter "G"
denotes deoxyguanosine, unless otherwise noted herein. The
oligonucleotides of the present invention can include non-natural
nucleotide analogs. However, where, for example, processing by
enzymes is required, or the like, oligonucleotides comprising
naturally occurring nucleotides are generally required for
maintenance of biological function.
Linking Primers to Solid Substrates
[0137] Anchor primers are linked to the solid substrate at the
sensitized sites. A region of a solid substrate containing a linked
primer is referred to herein as an anchor pad. Thus, by specifying
the sensitized states on the solid support, it is possible to form
an array or matrix of anchor pads. The anchor pads can be, e.g.,
small diameter spots etched at evenly spaced intervals on the solid
support. The anchor pads can be located at the bottoms of the
cavitations or wells if the substrate has been cavitated, etched,
or otherwise micromachined as discussed above.
[0138] In one embodiment, the anchor primer is linked to a
particle. The anchor primer can be linked to the particle prior to
formation of the extended anchor primer or after formation of the
extended anchor primer.
[0139] The anchor primer can be attached to the solid support via a
covalent or non-covalent interaction. In general, any linkage
recognized in the art can be used. Examples of such linkages common
in the art include any suitable metal (e.g., Co.sup.2+,
Ni.sup.2+)-hexahistidine complex, a biotin binding protein, e.g.,
NEUTRAVIDIN.TM. modified avidin (Pierce Chemicals, Rockford, Ill.),
streptavidin/biotin, avidin/biotin, glutathione S-transferase
(GST)/glutathione, monoclonal antibody/antigen, and maltose binding
protein/maltose, and pluronic coupling technologies. Samples
containing the appropriate tag are incubated with the sensitized
substrate so that zero, one, or multiple molecules attach at each
sensitized site.
[0140] One biotin-(strept-)avidin-based anchoring method uses a
thin layer of a photoactivatable biotin analog dried onto a solid
surface. (Hengsakul and Cass, 1996. Bioconjugate Chem. 7: 249-254).
The biotin analog is then exposed to white light through a mask, so
as to create defined areas of activated biotin. Avidin (or
streptavidin) is then added and allowed to bind to the activated
biotin. The avidin possesses free biotin binding sites which can be
utilized to "anchor" the biotinylated oligonucleotides through a
biotin-(strept-)avidin linkage.
[0141] Alternatively, the anchor primer can be attached to the
solid support with a biotin derivative possessing a photo-removable
protecting group. This moiety is covalently bound to bovine serum
albumin (BSA), which is attached to the solid support, e.g., a
glass surface. See Pirrung and Huang, 1996. Bioconjugate Chem. 7:
317-321. A mask is then used to create activated biotin within the
defined irradiated areas. Avidin may then be localized to the
irradiated area, with biotinylated DNA subsequently attached
through a BSA-biotin-avidin-biotin link. If desired, an
intermediate layer of silane is deposited in a self-assembled
monolayer on a silicon dioxide silane surface that can be patterned
to localize BSA binding in defined regions. See e.g., Mooney, et
al., 1996. Proc. Natl. Acad. Sci. USA 93: 12287-12291.
[0142] In pluronic based attachment, the anchor primers are first
attached to the termini of a polyethylene oxide-polypropylene
oxide-polyethylene oxide triblock copolymer, which is also known as
a pluronic compound. The pluronic moiety can be used to attach the
anchor primers to a solid substrate. Pluronics attach to
hydrophobic surfaces by virtue of the reaction between the
hydrophobic surface and the polypropylene oxide. The remaining
polyethylene oxide groups extend off the surface, thereby creating
a hydrophilic environment. Nitrilotriacetic acid (NTA) can be
conjugated to the terminal ends of the polyethylene oxide chains to
allow for hexahistidine tagged anchor primers to be attached. In
another embodiment, pyridyl disulfide (PDS) can be conjugated to
the ends of the polyethylene chains allowing for attachment of a
thiolated anchor primer via a disulfide bond. In one preferred
embodiment, Pluronic F108 (BASF Corp.) is used for the
attachment.
[0143] Each sensitized site on a solid support is potentially
capable of attaching multiple anchor primers. Thus, each anchor pad
may include one or more anchor primers. It is preferable to
maximize the number of pads that have only a single productive
reaction center (e.g., the number of pads that, after the extension
reaction, have only a single sequence extended from the anchor
primer). This can be accomplished by techniques which include, but
are not limited to: (i) varying the dilution of biotinylated anchor
primers that are washed over the surface; (ii) varying the
incubation time that the biotinylated primers are in contact with
the avidin surface; (iii) varying the concentration of open- or
closed-circular template so that, on average, only one primer on
each pad is extended to generate the sequencing template; or (iv)
reducing the size of the anchor pad to approach single-molecule
dimensions (<1 .mu.m) such that binding of one anchor inhibits
or blocks the binding of another anchor (e.g. by photoactivation of
a small spot); or (v) reducing the size of the anchor pad such that
binding of one circular template inhibits or blocks the binding of
a second circular template.
[0144] In some embodiments, each individual pad contains just one
linked anchor primer. Pads having only one anchor primer can be
made by performing limiting dilutions of a selected anchor primer
on to the solid support such that, on average, only one anchor
primer is deposited on each pad. The concentration of anchor primer
to be applied to a pad can be calculated utilizing, for example, a
Poisson distribution model.
[0145] In order to maximize the number of reaction pads that
contain a single anchor primer, a series of dilution experiments
are performed in which a range of anchor primer concentrations or
circular template concentrations are varied. For highly dilute
concentrations of primers, primers and circular templates binding
to the same pad will be independent of each other, and a Poisson
distribution will characterize the number of anchor primers
extended on any one pad. Although there will be variability in the
number of primers that are actually extended, a maximum of 37% of
the pads will have a single extended anchor primer (the number of
pads with a single anchor oligonucleotide). This number can be
obtained as follows.
[0146] Let N.sub.p be the average number of anchor primers on a pad
and f be the probability that an anchor primer is extended with a
circular template. Then the average number of extended anchor
primers per pad is N.sub.pf, which is defined as the quantity a.
There will be variability in the number of primers that are
actually extended. In the low-concentration limit, primers and
circular templates binding to the same pad will be independent of
each other, and a Poisson distribution P(n) will characterize the
number of anchor primers n extended on any pad. This distribution
may be mathematically defined by: P(n)=(a.sup.n/n!)exp(-a), with
P(1)=a exp(-a). The probability P(1) assumes its maximum value
exp(-1) for a=1, with 37% of pads having a single extended anchor
primer.
[0147] A range of anchor primer concentrations and circular
template concentrations may be subsequently scanned to find a value
of N.sub.pf closest to 1. A preferable method to optimize this
distribution is to allow multiple anchor primers on each reaction
pad, but use a limiting dilution of circular template so that, on
average, only one primer on each pad is extended to generate the
sequencing template.
[0148] Alternatively, at low concentrations of anchor primers, at
most one anchor primer will likely be bound on each reaction pad. A
high concentration of circular template may be used so that each
primer is likely to be extended.
[0149] Where the reaction pads are arrayed on a planar surface or a
fiber optic array, the individual pads are approximately 10 .mu.m
on a side, with a 100 .mu.m spacing between adjacent pads. Hence,
on a 1 cm.sup.2 surface a total of approximately 10,000
microreactors could be deposited, and, according to the Poisson
distribution, approximately 3700 of these will contain a single
anchor primer. In certain embodiments, after the primer
oligonucleotide has been attached to the solid support, modified,
e.g., biotinylated, enzymes are deposited to bind to the remaining,
unused avidin binding sites on the surface.
[0150] In other embodiments multiple anchor primers are attached to
any one individual pad in an array. Limiting dilutions of a
plurality of circular nucleic acid templates (described in more
detail below) may be hybridized to the anchor primers so
immobilized such that, on average, only one primer on each pad is
hybridized to a nucleic acid template. Library concentrations to be
used may be calculated utilizing, for example, limiting dilutions
and a Poisson distribution model.
Nucleic Acid Templates
[0151] The nucleic acid templates that can be sequenced according
to the invention, e.g., a nucleic acid library, in general can
include open circular or closed circular nucleic acid molecules. A
"closed circle" is a covalently closed circular nucleic acid
molecule, e.g., a circular DNA or RNA molecule. An "open circle" is
a linear single-stranded nucleic acid molecule having a 5'
phosphate group and a 3' hydroxyl group. In one embodiment, the
single stranded nucleic acid contains at least 100 copies of
nucleic acid sequence, each copy covalently linked end to end. In
some embodiments, the open circle is formed in situ from a linear
double-stranded nucleic acid molecule. The ends of a given open
circle nucleic acid molecule can be ligated by DNA ligase.
Sequences at the 5' and 3' ends of the open circle molecule are
complementary to two regions of adjacent nucleotides in a second
nucleic acid molecule, e.g., an adapter region of an anchor primer,
or to two regions that are nearly adjoining in a second DNA
molecule. Thus, the ends of the open-circle molecule can be ligated
using DNA ligase, or extended by DNA polymerase in a gap-filling
reaction. Open circles are described in detail in Lizardi, U.S.
Pat. No. 5,854,033. An open circle can be converted to a closed
circle in the presence of a DNA ligase (for DNA) or RNA ligase
following, e.g., annealing of the open circle to an anchor
primer.
[0152] If desired, nucleic acid templates can be provided as
padlock probes. Padlock probes are linear oligonucleotides that
include target-complementary sequences located at each end, and
which are separated by a linker sequence. The linkers can be
ligated to ends of members of a library of nucleic acid sequences
that have been, e.g., physically sheared or digested with
restriction endonucleases. Upon hybridization to a target-sequence,
the 5'- and 3'-terminal regions of these linear oligonucleotides
are brought in juxtaposition. This juxtaposition allows the two
probe segments (if properly hybridized) to be covalently-bound by
enzymatic ligation (e.g., with T4 DNA ligase), thus converting the
probes to circularly-closed molecules which are catenated to the
specific target sequences (see e.g., Nilsson, et al., 1994. Science
265: 2085-2088). The resulting probes are suitable for the
simultaneous analysis of many gene sequences both due to their
specificity and selectivity for gene sequence variants (see e.g.,
Lizardi, et al., 1998. Nat. Genet. 19: 225-232; Nilsson, et al.,
1997. Natl. Genet. 16: 252-255) and due to the fact that the
resulting reaction products remain localized to the specific target
sequences. Moreover, intramolecular ligation of many different
probes is expected to be less susceptible to non-specific
cross-reactivity than multiplex PCR-based methodologies where
non-cognate pairs of primers can give rise to irrelevant
amplification products (see e.g., Landegren and Nilsson, 1997. Ann.
Med. 29: 585-590).
[0153] A starting library can be constructed comprising either
single-stranded or double-stranded nucleic acid molecules, provided
that the nucleic acid sequence includes a region that, if present
in the library, is available for annealing, or can be made
available for annealing, to an anchor primer sequence. For example,
when used as a template for rolling circle amplification, a region
of a double-stranded template needs to be at least transiently
single-stranded in order to act as a template for extension of the
anchor primer.
[0154] Library templates can include multiple elements, including,
but not limited to, one or more regions that are complementary to
the anchor primer. For example, the template libraries may include
a region complementary to a sequencing primer, a control nucleotide
region, and an insert sequence comprised of the sequencing template
to be subsequently characterized. As is explained in more detail
below, the control nucleotide region is used to calibrate the
relationship between the amount of byproduct and the number of
nucleotides incorporated. As utilized herein the term "complement"
refers to nucleotide sequences that are able to hybridize to a
specific nucleotide sequence to form a matched duplex.
[0155] In one embodiment, a library template includes: (i) two
distinct regions that are complementary to the anchor primer, (ii)
one region homologous to the sequencing primer, (iii) one optional
control nucleotide region, (iv) an insert sequence of, e.g.,
30-500, 50-200, or 60-100 nucleotides, that is to be sequenced. The
template can, of course, include two, three, or all four of these
features.
[0156] The template nucleic acid can be constructed from any source
of nucleic acid, e.g., any cell, tissue, or organism, and can be
generated by any art-recognized method. Suitable methods include,
e.g., sonication of genomic DNA and digestion with one or more
restriction endonucleases (RE) to generate fragments of a desired
range of lengths from an initial population of nucleic acid
molecules. Preferably, one or more of the restriction enzymes have
distinct four-base recognition sequences. Examples of such enzymes
include, e.g., Sau3A1, MspI, and TaqI. Preferably, the enzymes are
used in conjunction with anchor primers having regions containing
recognition sequences for the corresponding restriction enzymes. In
some embodiments, one or both of the adapter regions of the anchor
primers contain additional sequences adjoining known restriction
enzyme recognition sequences, thereby allowing for capture or
annealing to the anchor primer of specific restriction fragments of
interest to the anchor primer. In other embodiments, the
restriction enzyme is used with a type IIS restriction enzyme.
[0157] Alternatively, template libraries can be made by generating
a complementary DNA (cDNA) library from RNA, e.g., messenger RNA
(mRNA). The cDNA library can, if desired, be further processed with
restriction endonucleases to obtain a 3' end characteristic of a
specific RNA, internal fragments, or fragments including the 3' end
of the isolated RNA. Adapter regions in the anchor primer may be
complementary to a sequence of interest that is thought to occur in
the template library, e.g., a known or suspected sequence
polymorphism within a fragment generated by endonuclease
digestion.
[0158] In one embodiment, an indexing oligonucleotide can be
attached to members of a template library to allow for subsequent
correlation of a template nucleic acid with a population of nucleic
acids from which the template nucleic acid is derived. For example,
one or more samples of a starting DNA population can be fragmented
separately using any of the previously disclosed methods (e.g.,
restriction digestion, sonication). An indexing oligonucleotide
sequence specific for each sample is attached to, e.g., ligated to,
the termini of members of the fragmented population. The indexing
oligonucleotide can act as a region for circularization,
amplification and, optionally, sequencing, which permits it to be
used to index, or code, a nucleic acid so as to identify the
starting sample from which it is derived.
[0159] Distinct template libraries made with a plurality of
distinguishable indexing primers can be mixed together for
subsequent reactions. Determining the sequence of the member of the
library allows for the identification of a sequence corresponding
to the indexing oligonucleotide. Based on this information, the
origin of any given fragment can be inferred.
Annealing and Amplification of Primer-Template Nucleic Acid
Complexes
[0160] Libraries of nucleic acids are annealed to anchor primer
sequences using recognized techniques (see, e.g., Hatch, et al.,
1999. Genet. Anal. Biomol. Engineer. 15: 35-40; Kool, U.S. Pat. No.
5,714,320 and Lizardi, U.S. Pat. No. 5,854,033). In general, any
procedure for annealing the anchor primers to the template nucleic
acid sequences is suitable as long as it results in formation of
specific, i.e., perfect or nearly perfect, complementarity between
the adapter region or regions in the anchor primer sequence and a
sequence present in the template library.
[0161] A number of in vitro nucleic acid amplification techniques
may be utilized to extend the anchor primer sequence. The size of
the amplified DNA preferably is smaller than the size of the anchor
pad and also smaller than the distance between anchor pads.
[0162] The amplification is typically performed in the presence of
a polymerase, e.g., a DNA or RNA-directed DNA polymerase, and one,
two, three, or four types of nucleotide triphosphates, and,
optionally, auxiliary binding proteins. In general, any polymerase
capable of extending a primed 3'-OH group can be used a long as it
lacks a 3' to 5' exonuclease activity. Suitable polymerases
include, e.g., the DNA polymerases from Bacillus
stearothermophilus, Thermus acquaticus, Pyrococcus furiosis,
Thermococcus litoralis, and Thermus thermophilus, bacteriophage T4
and T7, and the E. coli DNA polymerase I Klenow fragment. Suitable
RNA-directed DNA polymerases include, e.g., the reverse
transcriptase from the Avian Myeloblastosis Virus, the reverse
transcriptase from the Moloney Murine Leukemia Virus, and the
reverse transcriptase from the Human Immunodeficiency Virus-1.
[0163] A number of in vitro nucleic acid amplification techniques
have been described. These amplification methodologies may be
differentiated into those methods: (i) which require temperature
cycling-polymerase chain reaction (PCR) (see e.g., Saiki, et al.,
1995. Science 230: 1350-1354), ligase chain reaction (see e.g.,
Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189-193; Barringer, et
al., 1990. Gene 89: 117-122) and transcription-based amplification
(see e.g., Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86:
1173-1177) and (ii) isothermal amplification
systems--self-sustaining, sequence replication (see e.g., Guatelli,
et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878); the
Q.beta. replicase system (see e.g., Lizardi, et al., 1988.
BioTechnology 6: 1197-1202); strand displacement amplification
Nucleic Acids Res. Apr. 11, 1992; 20(7):1691-6.; and the methods
described in PNAS Jan. 1, 1992; 89(1):392-6; and NASBA J Virol
Methods. December 1991; 35(3):273-86.
[0164] Isothermal amplification also includes rolling circle-based
amplification (RCA). RCA is discussed in, e.g., Kool, U.S. Pat. No.
5,714,320 and Lizardi, U.S. Pat. No. 5,854,033; Hatch, et al.,
1999. Genet. Anal Biomol. Engineer. 15: 35-40. The result of the
RCA is a single DNA strand extended from the 3' terminus of the
anchor primer (and thus is linked to the solid support matrix) and
including a concatamer containing multiple copies of the circular
template annealed to a primer sequence. Typically, 1,000 to 10,000
or more copies of circular templates, each having a size of, e.g.,
approximately 30-500, 50-200, or 60-100 nucleotides size range, can
be obtained with RCA.
[0165] The product of RCA amplification following annealing of a
circular nucleic acid molecule to an anchor primer is shown
schematically in FIG. 1A. A circular template nucleic acid 102 is
annealed to an anchor primer 104, which has been linked to a
surface 106 at its 5' end and has a free 3' OH available for
extension. The circular template nucleic acid 102 includes two
adapter regions 108 and 110 which are complementary to regions of
sequence in the anchor primer 104. Also included in the circular
template nucleic acid 102 is an insert 112 and a region 114
homologous to a sequencing primer, which is used in the sequencing
reactions described below.
[0166] Upon annealing, the free 3'-OH on the anchor primer 104 can
be extended using sequences within the template nucleic acid 102.
The anchor primer 102 can be extended along the template multiple
times, with each iteration adding to the sequence extended from the
anchor primer a sequence complementary to the circular template
nucleic acid. Four iterations, or four rounds of rolling circle
replication, are shown in FIG. 1A as the extended anchor primer
amplification product 114. Extension of the anchor primer results
in an amplification product covalently or otherwise physically
attached to the substrate 106.
[0167] Additional embodiments of circular templates and anchor
primers are shown in more detail in FIGS. 1B-1D. FIG. 1B
illustrates an annealed open circle linear substrate that can
serve, upon ligation, as a template for extension of an anchor
primer. A template molecule having the sequence 5'-TCg TgT gAg gTC
TCA gCA TCT TAT gTA TAT TTA CTT CTA TTC TCA gTT gCC TAA gCT gCA gCC
A-3' (SEQ ID NO:1) is annealed to an anchor primer having a biotin
linker at its 5' terminus and the sequence 5'-gAC CTC ACA CgA Tgg
CTg CAg CTT-3' (SEQ ID NO:2). Annealing of the template results in
juxtaposition of the 5' and 3' ends of the template molecule. The
3'OH of the anchor primer can be extended using the circular
template.
[0168] The use of a circular template and an anchor primer for
identification of single nucleotide polymorphisms is shown in FIG.
1C. Shown is a generic anchor primer having the sequence 5'-gAC CTC
ACA CgA Tgg CTg CAg CTT-3' (SEQ ID NO:3). The anchor primer anneals
to an SNP probe having the sequence 5'-TTT ATA TgT ATT CTA CgA CTC
Tgg AgT gTg CTA CCg ACg TCg AAt CCg TTg ACT CTT ATC TTC A-3' (SEQ
ID NO:4). The SNP probe in turn hybridizes to a region of a
SNP-containing region of a gene having the sequence 5'-CTA gCT CgT
ACA TAT AAA TgA AgA TAA gAT CCT g -3' (SEQ ID NO:5). Hybridization
of a nucleic acid sequence containing the polymorphism to the SNP
probe complex allows for subsequent ligation and circularization of
the SNP probe. The SNP probe is designed so that its 5' and 3'
termini anneal to the genomic region so as to abut in the region of
the polymorphic site, as is indicated in FIG. 1C. The circularized
SNP probe can be subsequently extended and sequenced using the
methods described herein. A nucleic acid lacking the polymorphism
does not hybridize so as to result in juxtaposition of the 5' and
3' termini of the SNP probe. In this case, the SNP probe cannot be
ligated to form a circular substrate needed for subsequent
extension.
[0169] FIG. 1D illustrates the use of a gap oligonucleotide to
along with a circular template molecule. An anchor primer having
the sequence 5'-gAC CTC ACA CgA gTA gCA Tgg CTg CAg CTT-3' (SEQ ID
NO:6) is attached to a surface through a biotin linker. A template
molecule having the sequence 5'-TCg TgT gAg gTC TCA gCA TCT TAT gTA
TAT TTA CTT CTA TTC TCA gTT gCC TAA gCT gCA gCC A-3' (SEQ ID NO:7)
is annealed to the anchor primer to result in partially single
stranded, or gapped region, in the anchor primer flanked by a
double-stranded region. A gapping molecule having the sequence
5'-TgC TAC-3' then anneals to the anchor primer. Ligation of both
ends of the gap oligonucleotide to the template molecule results in
formation of a circular nucleic acid molecule that can act as a
template for rolling circle amplification.
[0170] Circular oligonucleotides that are generated during
polymerase-mediated DNA replication are dependent upon the
relationship between the template and the site of replication
initiation. In double-stranded DNA templates, the critical features
include whether the template is linear or circular in nature, and
whether the site of initiation of replication (i.e., the
replication "fork") is engaged in synthesizing both strands of DNA
or only one. In conventional double-stranded DNA replication, the
replication fork is treated as the site at which the new strands of
DNA are synthesized. However, in linear molecules (whether
replicated unidirectionally or bidirectionally), the movement of
the replication fork(s) generate a specific type of structural
motif. If the template is circular, one possible spatial
orientation of the replicating molecule takes the form of a .theta.
structure.
[0171] Alternatively, RCA can occur when the replication of the
duplex molecule begins at the origin. Subsequently, a nick opens
one of the strands, and the free 3'-terminal hydroxyl moiety
generated by the nick is extended by the action of DNA polymerase.
The newly synthesized strand eventually displaces the original
parental DNA strand. This aforementioned type of replication is
known as rolling-circle replication (RCR) because the point of
replication may be envisaged as "rolling around" the circular
template strand and, theoretically, it could continue to do so
indefinitely. Additionally, because the newly synthesized DNA
strand is covalently-bound to the original template, the displaced
strand possesses the original genomic sequence (e.g., gene or other
sequence of interest) at its 5'-terminus. In RCR, the original
genomic sequence is followed by any number of "replication units"
complementary to the original template sequence, wherein each
replication unit is synthesized by continuing revolutions of said
original template sequence. Hence, each subsequent revolution
displaces the DNA which is synthesized in the previous replication
cycle.
[0172] In vivo, RCR is utilized in several biological systems. For
example, the genome of several bacteriophage are single-stranded,
circular DNA. During replication, the circular DNA is initially
converted to a duplex form, which is then replicated by the
aforementioned rolling-circle replication mechanism. The displaced
terminus generates a series of genomic units that can be cleaved
and inserted into the phage particles. Additionally, the displaced
single-strand of a rolling-circle can be converted to duplex DNA by
synthesis of a complementary DNA strand. This synthesis can be used
to generate the concatemeric duplex molecules required for the
maturation of certain phage DNAs. For example, this provides the
principle pathway by which .lamda. bacteriophage matures. RCR is
also used in vivo to generate amplified rDNA in Xenopus oocytes,
and this fact may help explain why the amplified rDNA is comprised
of a large number of identical repeating units. In this case, a
single genomic repeating unit is converted into a rolling-circle.
The displaced terminus is then converted into duplex DNA which is
subsequently cleaved from the circle so that the two termini can be
ligated together so as to generate the amplified circle of
rDNA.
[0173] Through the use of the RCA reaction, a strand may be
generated which represents many tandem copies of the complement to
the circularized molecule. For example, RCA has recently been
utilized to obtain an isothermal cascade amplification reaction of
circularized padlock probes in vitro in order to detect single-copy
genes in human genomic DNA samples (see Lizardi, et al., 1998. Nat.
Genet. 19: 225-232). In addition, RCA has also been utilized to
detect single DNA molecules in a solid phase-based assay, although
difficulties arose when this technique was applied to in situ
hybridization (see Lizardi, et al., 1998. Nat. Genet. 19:
225-232).
[0174] If desired, RCA can be performed at elevated temperatures,
e.g., at temperatures greater than 37.degree. C., 42.degree. C.,
45.degree. C., 50.degree. C., 60.degree. C., or 70.degree. C. In
addition, RCA can be performed initially at a lower temperature,
e.g., room temperature, and then shifted to an elevated
temperature. Elevated temperature RCA is preferably performed with
thermostable nucleic acid polymerases and with primers that can
anneal stably and with specificity at elevated temperatures.
[0175] RCA can also be performed with non-naturally occurring
oligonucleotides, e.g., peptide nucleic acids. Further, RCA can be
performed in the presence of auxiliary proteins such as
single-stranded binding proteins.
[0176] The development of a method of amplifying short DNA
molecules which have been immobilized to a solid support, termed
RCA has been recently described in the literature (see e.g., Hatch,
et al., 1999. Genet. Anal. Biomol. Engineer. 15: 3540; Zhang, et
al., 1998. Gene 211: 277-85; Baner, et al., 1998. Nucl. Acids Res.
26: 5073-5078; Liu, et al., 1995. J. Am. Chem. Soc. 118: 1587-1594;
Fire and Xu, 1995. Proc. Natl. Acad. Sci. USA 92: 4641-4645;
Nilsson, et al., 1994. Science 265: 2085-2088). RCA targets
specific DNA sequences through hybridization and a DNA ligase
reaction. The circular product is then subsequently used as a
template in a rolling circle replication reaction.
[0177] RCA driven by DNA polymerase can replicate circularized
oligonucleotide probes with either linear or geometric kinetics
under isothermal conditions. In the presence of two primers (one
hybridizing to the + strand, and the other, to the - strand of
DNA), a complex pattern of DNA strand displacement ensues which
possesses the ability to generate 1.times.10.sup.9 or more copies
of each circle in a short period of time (i.e., less-than 90
minutes), enabling the detection of single-point mutations within
the human genome. Using a single primer, RCA generates hundreds of
randomly-linked copies of a covalently closed circle in several
minutes. If solid support matrix-associated, the DNA product
remains bound at the site of synthesis, where it may be labeled,
condensed, and imaged as a point light source. For example, linear
oligonucleotide probes, which can generate RCA signals, have been
bound covalently onto a glass surface. The color of the signal
generated by these probes indicates the allele status of the
target, depending upon the outcome of specific, target-directed
ligation events. As RCA permits millions of individual probe
molecules to be counted and sorted, it is particularly amenable for
the analysis of rare somatic mutations. RCA also shows promise for
the detection of padlock probes bound to single-copy genes in
cytological preparations.
[0178] In addition, a solid-phase RCA methodology has also been
developed to provide an effective method of detecting constituents
within a solution. Initially, a recognition step is used to
generate a complex h a circular template is bound to a surface. A
polymerase enzyme is then used to amplify the bound complex. RCA
uses small DNA probes that are amplified to provide an intense
signal using detection methods, including the methods described in
more detail below.
[0179] Other examples of isothermal amplification systems include,
e.g., (i) self-sustaining, sequence replication (see e.g.,
Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878),
(ii) the Q.beta. replicase system (see e.g., Lizardi, et al., 1988.
BioTechnology 6: 1197-1202), and (iii) nucleic acid sequence-based
amplification (NASBA.TM.; see Kievits, et al., 1991. J. Virol.
Methods 35: 273-286).
Methods for Determining the Nucleotide Sequence of the Amplified
Product
[0180] Amplification of a nucleic acid template as described above
results in multiple copies of a template nucleic acid sequence
covalently linked to an anchor primer. In one embodiment, a region
of the sequence product is determined by annealing a sequencing
primer to a region of the template nucleic acid, and then
contacting the sequencing primer with a DNA polymerase and a known
nucleotide triphosphate, i.e., dATP, dCTP, dGTP, dTFP, or an analog
of one of these nucleotides. The sequence can be determined by
detecting a sequence reaction byproduct, as is described below.
[0181] The sequence primer can be any length or base composition,
as long as it is capable of specifically annealing to a region of
the amplified nucleic acid template. No particular structure for
the sequencing primer is required so long as it is able to
specifically prime a region on the amplified template nucleic acid.
Preferably, the sequencing primer is complementary to a region of
the template that is between the sequence to be characterized and
the sequence hybridizable to the anchor primer. The sequencing
primer is extended with the DNA polymerase to form a sequence
product. The extension is performed in the presence of one or more
types of nucleotide triphosphates, and if desired, auxiliary
binding proteins.
[0182] Incorporation of the dNTP is preferably determined by
assaying for the presence of a sequencing byproduct. In a preferred
embodiment, the nucleotide sequence of the sequencing product is
determined by measuring inorganic pyrophosphate (PPi) liberated
from a nucleotide triphosphate (dNTP) as the dNMP is incorporated
into an extended sequence primer. This method of sequencing, termed
Pyrosequencing.TM. technology (PyroSequencing AB, Stockholm,
Sweden) can be performed in solution (liquid phase) or as a solid
phase technique. PPi-based sequencing methods are described
generally in, e.g., WO9813523A1, Ronaghi, et al., 1996. Anal.
Biochem. 242: 84-89, and Ronaghi, et al., 1998. Science 281:
363-365 (1998). These disclosures of PPi sequencing are
incorporated herein in their entirety, by reference.
[0183] Pyrophosphate released under these conditions can be
detected enzymatically (e.g., by the generation of light in the
luciferase-luciferin reaction). Such methods enable a nucleotide to
be identified in a given target position, and the DNA to be
sequenced simply and rapidly while avoiding the need for
electrophoresis and the use of potentially dangerous
radiolabels.
[0184] PPi can be detected by a number of different methodologies,
and various enzymatic methods have been previously described (see
e.g., Reeves, et al., 1969. Anal. Biochem. 28: 282-287; Guillory,
et al, 1971. Anal. Biochem. 39: 170-180; Johnson, et al., 1968.
Anal. Biochem. 15: 273; Cook, et al., 1978. Anal. Biochem. 91:
557-565; and Drake, et al., 1979. Anal. Biochem. 94: 117-120).
[0185] PPi liberated as a result of incorporation of a dNTP by a
polymerase can be converted to ATP using, e.g., an ATP sulfurylase.
This enzyme has been identified as being involved in sulfur
metabolism. Sulfur, in both reduced and oxidized forms, is an
essential mineral nutrient for plant and animal growth (see e.g.,
Schmidt and Jager, 1992. Ann. Rev. Plant Physiol. Plant Mol. Biol.
43: 325-349). In both plants and microorganisms, active uptake of
sulfate is followed by reduction to sulfide. As sulfate has a very
low oxidation/reduction potential relative to available cellular
reductants, the primary step in assimilation requires its
activation via an ATP-dependent reaction (see e.g., Leyh, 1993.
Crit. Rev. Biochem. Mol. Biol. 28: 515-542). ATP sulfurylase (ATP:
sulfate adenylyltransferase; EC 2.7.7.4) catalyzes the initial
reaction in the metabolism of inorganic sulfate (SO.sub.4.sup.-2);
see e.g., Robbins and Lipmann, 1958. J. Biol. Chem. 233: 686-690;
Hawes and Nicholas, 1973. Biochem. J. 133: 541-550). In this
reaction SO.sub.4.sup.-2 is activated to adenosine
5'-phosphosulfate (APS).
[0186] ATP sulfurylase has been highly purified from several
sources such as Saccharomyces cereisiae (see e.g., Hawes and
Nicholas, 1973. Biochem. J. 133: 541-550); Penicillium chrysogenum
(see e.g., Renosto, et al., 1990. J. Biol. Chem. 265: 10300-10308);
rat liver (see e.g.. Yu. et al., 1989. Arch. Biochem. Biophys. 269:
165-174); and plants (see e.g., Shaw and Anderson, 1972. Biochem.
J. 127: 237-247; Osslund, et al, 1982. Plant Physiol. 70: 39-45).
Furthermore, ATP sulfurylase genes have been cloned from
prokaryotes (see e.g., Leyh, et al., 1992. J. Biol. Chem. 267:
10405-10410: Schwedock and Long, 1989. Mol. Plant Microbe
Interaction 2: 181-194; Laue and Nelson, 1994. J. Bacteriol. 176:
3723-3729); eukaryotes (see e.g., Cherest, et al., 1987. Mol. Gen.
Genet. 210: 307-313; Mountain and Korch, 1991. Yeast 7: 873-880;
Foster, et al., 1994. J. Biol. Chem. 269: 19777-19786); plants (see
e.g., Leustek, et al., 1994. Plant Physiol. 105: 897-90216); and
animals (see e.g., Li, et al., 1995. J. Biol. Chem. 270:
29453-29459). The enzyme is a homo-oligomer or heterodimer,
depending upon the specific source (see e.g., Leyh and Suo, 1992.
J. Biol. Chem. 267: 542-545).
[0187] In some embodiments, a thermostable sulfurylase is used.
Thermostable sulfurylases can be obtained from, e.g., Archaeoglobus
or Pyrococcus spp. Sequences of thermostable sulfurylases are
available at database Acc. No. 028606, Acc. No. Q9YCR4, and Acc.
No. P56863.
[0188] ATP sulfurylase has been used for many different
applications, for example, bioluminometric detection of ADP at high
concentrations of ATP (see e.g., Schultz, et al., 1993. Anal.
Biochem. 215: 302-304); continuous monitoring of DNA polymerase
activity (see e.g., Nyrbn, 1987. Anal. Biochem. 167: 235-238); and
DNA sequencing (see e.g., Ronaghi, et al., 1996. Anal. Biochem.
242: 84-89; Ronaghi, et al., 1998. Science 281: 363-365; Ronaghi,
et al., 1998. Anal. Biochem. 267: 65-71).
[0189] Several assays have been developed for detection of the
forward ATP sulfurylase reaction. The colorimetric molybdolysis
assay is based on phosphate detection (see e.g., Wilson and
Bandurski, 1958. J. Biol. Chem. 233: 975-981), whereas the
continuous spectrophotometric molybdolysis assay is based upon the
detection of NADH oxidation (see e.g., Seubert, et al., 1983. Arch.
Biochem. Biophys. 225: 679-691; Seubert, et al., 1985. Arch.
Biochem. Biophys. 240: 509-523). The later assay requires the
presence of several detection enzymes. In addition, several
radioactive assays have also been described in the literature (see
e.g., Daley, et al., 1986. Anal. Biochem. 157: 385-395). For
example, one assay is based upon the detection of .sup.32PPi
released from .sup.32P-labeled ATP (see e.g., Scubert, et al.,
1985. Arch. Biochem. Biophys. 240: 509-523) and another on the
incorporation of .sup.35S into [.sup.35S]-labeled APS (this assay
also requires purified APS kinase as a coupling enzyme; see e.g.,
Scubert, et al., 1983. Arch. Biochem. Biophys. 225: 679-691); and a
third reaction depends upon the release of .sup.35SO.sub.4.sup.-2
from [.sup.35S]-labeled APS (see e.g., Daley, et al., 1986. Anal.
Biochem. 157: 385-395).
[0190] For detection of the reversed ATP sulfurylase reaction a
continuous spectrophotometric assay (see e.g., Segel, et al., 1987.
Methods Enzymol. 143: 334-349); a bioluminometric assay (see e.g.,
Balharry and Nicholas, 1971. Anal. Biochem. 40: 1-17); an
.sup.35SO.sub.4.sup.-2 release assay (see e.g., Seubert, et al.,
1985. Arch. Biochem. Biophys. 240: 509-523); and a .sup.32PPi
incorporation assay (see e.g., Osslund, et al., 1982. Plant
Physiol. 70: 39-45) have been previously described.
[0191] ATP produced by an ATP sulfurylase can be hydrolyzed using
enzymatic reactions to generate light. Light-emitting chemical
reactions (i.e., chemiluminescence) and biological reactions (i.e.,
bioluminescence) are widely used in analytical biochemistry for
sensitive measurements of various metabolites. In bioluminescent
reactions, the chemical reaction that leads to the emission of
light is enzyme-catalyzed. For example, the luciferin-luciferase
system allows for specific assay of ATP and the bacterial
luciferase-oxidoreductase system can be used for monitoring of
NAD(P)H. Both systems have been extended to the analysis of
numerous substances by means of coupled reactions involving the
production or utilization of ATP or NAD(P)H (see e.g., Kricka,
1991. Chemiluminescent and bioluminescent techniques. Clin. Chem.
37: 1472-1281).
[0192] The development of new reagents have made it possible to
obtain stable light emission proportional to the concentrations of
ATP (see e.g., Lundin, 1982. Applications of firefly luciferase In;
Luminescent Assays (Raven Press, New York) or NAD(P)H (see e.g.,
Lovgren, et al., Continuous monitoring of NADH-converting reactions
by bacterial luminescence. J. Appl. Biochem. 4: 103-111). With such
stable light emission reagents, it is possible to make endpoint
assays and to calibrate each individual assay by addition of a
known amount of ATP or NAD(P)H. In addition, a stable
light-emitting system also allows continuous monitoring of ATP- or
NAD(P)H-converting systems.
[0193] Suitable enzymes for converting ATP into light include
luciferases, e.g., insect luciferases. Luciferases produce light as
an end-product of catalysis. The best known light-emitting enzyme
is that of the firefly, Photinus pyralis (Coleoptera). The
corresponding gene has been cloned and expressed in bacteria (see
e.g., de Wet, et al., 1985. Proc. Natl. Acad. Sci. USA 80:
7870-7873) and plants (see e.g., Ow, et al., 1986. Science 234:
856-859), as well as in insect (see e.g., Jha, et al., 1990. FEBS
Lett. 274: 24-26) and mammalian cells (see e.g., de Wet, et al.,
1987. Mol. Cell. Biol. 7: 725-7373; Keller, et al., 1987. Proc.
Natl. Acad. Sci. USA 82: 3264-3268). In addition, a number of
luciferase genes from the Jamaican click beetle Pyroplorus
plagiophihalamus (Coleoptera), have recently been cloned and
partially characterized (see e.g., Wood, et al., 1989. J. Biolumin.
Chemilumin. 4: 289-301; Wood, et al., 1989. Science 244: 700-702).
Distinct luciferases can sometimes produce light of different
wavelengths, which may enable simultaneous monitoring of light
emissions at different wavelengths. Accordingly, these
aforementioned characteristics are unique, and add new dimensions
with respect to the utilization of current reporter systems.
[0194] Firefly luciferase catalyzes bioluminescence in the presence
of luciferin, adenosine 5'-triphosphate (ATP), magnesium ions, and
oxygen, resulting in a quantum yield of 0.88 (see e.g., McElroy and
Selinger, 1960. Arch. Biochem. Biophys. 88: 136-145). The firefly
luciferase bioluminescent reaction can be utilized as an assay for
the detection of ATP with a detection limit of approximately
1.times.10.sup.-13 M (see e.g., Leach, 1981. J. Appl. Biochem. 3:
473-517). In addition, the overall degree of sensitivity and
convenience of the luciferase-mediated detection systems have
created considerable interest in the development of firefly
luciferase-based biosensors (see e.g., Green and Kricka, 1984.
Talanta 31: 173-176; Blum, et al., 1989. J. Biolumin. Chemilumin.
4: 543-550).
[0195] Using the above-described enzymes, the sequence primer is
exposed to a polymerase and a known dNTP. If the dNTP is
incorporated onto the 3' end of the primer sequence, the dNTP is
cleaved and a PPi molecule is liberated. The PPi is then converted
to ATP with ATP sulfurylase. Preferably, the ATP sulfurylase is
present at a sufficiently high concentration that the conversion of
PPi proceeds with first-order kinetics with respect to PPi. In the
presence of luciferase, the ATP is hydrolyzed to generate a photon.
The reaction preferably has a sufficient concentration of
luciferase present within the reaction mixture such that the
reaction, ATP.fwdarw.ADP+PO.sub.4.sup.3-+photon (light), proceeds
with first-order kinetics with respect to ATP. The photon can be
measured using methods and apparatuses described below. In one
embodiment, the PPi and a coupled sulfurylase/luciferase reaction
is used to generate light for detection. In some embodiments,
either or both the sulfurylase and luciferase are immobilized on
one or more mobile solid supports disposed at each reaction
site.
[0196] The present invention thus permits PPi release to be
detected during the polymerase reaction giving a real-time signal.
The sequencing reactions may be continuously monitored in
real-time. A procedure for rapid detection of PPi release is thus
enabled by the present invention. The reactions have been estimated
to take place in less than 2 seconds (Nyren and Lundin, supra). The
rate limiting step is the conversion of PPi to ATP by ATP
sulfurylase, while the luciferase reaction is fast and has been
estimated to take less than 0.2 seconds Incorporation rates for
polymerases have also been estimated by various methods and it has
been found, for example, that in the case of Klenow polymerase,
complete incorporation of one base may take less than 0.5 seconds.
Thus, the estimated total time for incorporation of one base and
detection by this enzymatic assay is approximately 3 seconds. It
will be seen therefore that very fast reaction times are possible,
enabling real-time detection. The reaction times could further be
decreased by using a more thermostable luciferase.
[0197] For most applications it is desirable to use reagents free
of contaminants like ATP and PPi. These contaminants may be removed
by flowing the reagents through a pre-column containing apyrase
and/-or pyrophosphatase bound to resin. Alternatively, the apyrase
or pyrophosphatase can be bound to magnetic beads and used to
remove contaminating ATP and PPi present in the reagents. In
addition it is desirable to wash away diffusible sequencing
reagents, e.g., unincorporated dNTPs, with a wash buffer. Any wash
buffer used in pyrophosphate sequencing can be used.
[0198] In some embodiments, the concentration of reactants in the
sequencing reaction include 1 pmol DNA, 3 pmol polymerase, 40 pmol
dNTP in 0.2 ml buffer. See Ronaghi, et al., Anal. Biochem. 242:
84-89 (1996).
[0199] The sequencing reaction can be performed with each of four
predetermined nucleotides, if desired. A "complete" cycle generally
includes sequentially administering sequencing reagents for each of
the nucleotides dATP, dGTP, dCTP and dTTP (or dUTP), in a
predetermined order. Unincorporated dNTPs are washed away between
each of the nucleotide additions. Alternatively, unincorporated
dNTPs are degraded by apyrase (see below). The cycle is repeated as
desired until the desired amount of sequence of the sequence
product is obtained. In some embodiments, about 10-1000, 10-100,
10-75, 20-50, or about 30 nucleotides of sequence information is
obtained from extension of one annealed sequencing primer.
[0200] In some embodiments, the nucleotide is modified to contain a
disulfide-derivative of a hapten such as biotin. The addition of
the modified nucleotide to the nascent primer annealed to the
anchored substrate is analyzed by a post-polymerization step that
includes i) sequentially binding of, in the example where the
modification is a biotin, an avidin- or streptavidin-conjugated
moiety linked to an enzyme molecule, ii) the washing away of excess
avidin- or streptavidin-linked enzyme, iii) the flow of a suitable
enzyme substrate under conditions amenable to enzyme activity, and
iv) the detection of enzyme substrate reaction product or products.
The hapten is removed in this embodiment through the addition of a
reducing agent. Such methods enable a nucleotide to be identified
in a given target position, and the DNA to be sequenced simply and
rapidly while avoiding the need for electrophoresis and the use of
potentially dangerous radiolabels.
[0201] A preferred enzyme for detecting the hapten is horse-radish
peroxidase. If desired, a wash buffer, can be used between the
addition of various reactants herein. Apyrase can be used to remove
unreacted dNTP used to extend the sequencing primer. The wash
buffer can optionally include apyrase.
[0202] Example haptens, e.g., biotin, digoxygenin, the fluorescent
dye molecules cy3 and cy5, and fluorescein, are incorporated at
various efficiencies into extended DNA molecules. The attachment of
the hapten can occur through linkages via the sugar, the base, and
via the phosphate moiety on the nucleotide. Example means for
signal amplification include fluorescent, electrochemical and
enzymatic. In a preferred embodiment using enzymatic amplification,
the enzyme, e.g. alkaline phosphatase (AP), horse-radish peroxidase
(HRP), beta-galactosidase, luciferase, can include those for which
light-generating substrates are known, and the means for detection
of these light-generating (chemiluminescent) substrates can include
a CCD camera.
[0203] In a preferred mode, the modified base is added, detection
occurs, and the hapten-conjugated moiety is removed or inactivated
by use of either a cleaving or inactivating agent. For example, if
the cleavable-linker is a disulfide, then the cleaving agent can be
a reducing agent, for example dithiothreitol (DTT),
beta-mercaptoethanol, etc. Other embodiments of inactivation
include heat, cold, chemical denaturants, surfactants, hydrophobic
reagents, and suicide inhibitors.
[0204] Luciferase can hydrolyze dATP directly with concomitant
release of a photon. This results in a false positive signal
because the hydrolysis occurs independent of incorporation of the
dATP into the extended sequencing primer. To avoid this problem, a
dATP analog can be used which is incorporated into DNA, i.e., it is
a substrate for a DNA polymerase, but is not a substrate for
luciferase. One such analog is .alpha.-thio-dATP. Thus, use of
.alpha.-thio-dATP avoids the spurious photon generation that can
occur when dATP is hydrolyzed without being incorporated into a
growing nucleic acid chain.
[0205] Typically, the PPi-based detection is calibrated by the
measurement of the light released following the addition of control
nucleotides to the sequencing reaction mixture immediately after
the addition of the sequencing primer. This allows for
normalization of the reaction conditions. Incorporation of two or
more identical nucleotides in succession is revealed by a
corresponding increase in the amount of light released. Thus, a
two-fold increase in released light relative to control nucleotides
reveals the incorporation of two successive dNTPs into the extended
primer.
[0206] If desired, apyrase may be "washed" or "flowed" over the
surface of the solid support so as to facilitate the degradation of
any remaining, non-incorporated dNTPs within the sequencing
reaction mixture. Apyrase also degrades the generated ATP and hence
"turns off" the light generated from the reaction. Upon treatment
with apyrase, any remaining reactants are washed away in
preparation for the following dNTP incubation and photon detection
steps. Alternatively, the apyrase may be bound to the solid or
mobile solid support.
[0207] When the support is planar, the pyrophosphate sequencing
reactions preferably take place in a thin reaction chamber that
includes one optically transparent solid support surface and an
optically transparent cover. In some embodiments, the array has a
planar top surface and a planar bottom surface, the planar top
surface has at least 1,000 cavities thereon each cavity forming a
reaction chamber. In additional embodiments, the planar bottom
surface is optically conductive such that optical signals from the
reaction chambers can be detected through the bottom planar
surface. In a preferred embodiment, the distance between the top
surface and the bottom surface is no greater than 10 cm. Sequencing
reagents may then be delivered by flowing them across the surface
of the substrate. More preferably, the cavities contain reagents
for analyzing a nucleic acid or protein. In an additional
embodiment, the array has a second surface spaced apart from the
planar array and in opposing contact therewith such that a flow
chamber is formed over the array. When the support is not planar,
the reagents may be delivered by dipping the solid support into
baths of any given reagents.
[0208] In a preferred embodiment, an array can be used to carry out
separate parallel common reactions in an aqueous environment. The
array can have a substrate having at least 1,000 discrete reaction
chambers containing a starting material that is capable of reacting
with a reagent, each of the reaction chambers being dimensioned
such that when one or more fluids containing at least one reagent
is delivered into each reaction chamber, the diffusion time for the
reagent to diffuse out of the well exceeds the time required for
the starting material to react with the reagent to form a product.
The reaction chambers can be formed by generating a plurality of
cavities on the substrate. The plurality of cavities can be formed
in the substrate via etching, molding or micromaching. The cavities
can have a planar bottom or a concave bottom. In a preferred
embodiment, the substrate is a fiber optic bundle. In an additional
embodiment, the reaction chambers are formed by generating discrete
patches on a planar surface. The patches can have a different
surface chemistry than the surrounding planar surface.
[0209] In various embodiments, some components of the reaction are
immobilized, while other components are provided in solution. For
example, in some embodiments, the enzymes utilized in the
pyrophosphate sequencing reaction (e.g., sulfurylase, luciferase)
may be immobilized if desired onto the solid support. Similarly,
one or more or of the enzymes utilized in the pyrophosphate
sequencing reaction, e.g., sulfurylase, luciferase may be
immobilized at the termini of a fiber optic reactor array. When
luciferase is immobilized, it is preferably less than 50 .mu.m from
an anchored primer. Other components of the reaction, e.g., a
polymerase (such as Klenow fragment), nucleic acid template, and
nucleotides can be added by flowing, spraying, or rolling. In still
further embodiments, one more of the reagents used in the
sequencing reactions is delivered on beads.
[0210] In some embodiments, reagents are dispensed using an
expandable, flexible membrane to dispense reagents and seal
reactors on FORA surface during extension reactions. Reagents can
be sprayed or rolled onto either the FORA surface or onto the
flexible membrane. The flexible membrane could then be either
rapidly expanded or physically moved into close proximity with the
FORA thereby sealing the wells such that PPi would be unable to
diffuse from well to well. Preferably, data acquisition takes place
at a reasonable time after reaction initiation to allow maximal
signal to generate.
[0211] A sequence in an extended anchor primer can also be
identified using sequencing methods other than by detecting a
sequence byproduct. For example, sequencing can be performed by
measuring incorporation of labeled nucleotides or other nucleotide
analogs. These methods can be used in conjunction with fluorescent
or electrochemiluminescent-based methods.
[0212] Alternatively, sequence byproducts can be generated using
dideoxynucleotides having a label on the 3' carbon. Preferably, the
label can be cleaved to reveal a 3' hydroxyl group. In this method,
addition of a given nucleotide is scored as positive or negative,
and one base is determined at each trial. In this embodiment, solid
phase enzymes are not required and multiple measurements can be
made.
[0213] In another embodiment, the identity of the extended anchor
primer product is determined using labeled deoxynucleotides. The
labeled deoxynucleotides can be, e.g., fluorescent nucleotides.
Preferably the fluorescent nucleolides can be detected following
laser-irradiation. Preferably, the fluorescent label is not stable
for long periods of exposure. If desired, the fluorescent signal
can be quenched, e.g., photobleached, to return signal to
background levels prior to addition of the next base. A preferred
electrochemiluminescent label is ruthenium-tris-bi-pyridyl.
[0214] In one embodiment, a single stranded circular nucleic acid
is immobilized in the reaction chamber; preferably each reaction
chamber has no more than one single stranded circular nucleic acid
disposed therein. More preferably, a single stranded circular
nucleic acid is immobilized on a mobile solid support disposed in
the reaction chamber. In another embodiment, each single stranded
circular nucleic acid contains at least 100 copies of a nucleic
acid sequence, each copy covalently linked end to end.
[0215] The invention also comprises kits for use in methods of the
invention which could include one or more of the following
components: (a) a test specific primer which hybridizes to sample
DNA so that the target position is directly adjacent to the 3' end
of the primer; (b) a polymerase; (c) detection enzyme means for
identifying PPi release; (d) deoxynucleotides including, in place
of dATP, a dATP analogue which is capable of acting as a substrate
for a polymerase but incapable of acting as a substrate for a said
PPi-detection enzyme; and (e) optionally dideoxynucleotides,
optionally ddATP being replaced by a ddATP analogue which is
capable of acting as a substrate for a polymerase but incapable of
acting as a substrate for a said PPi-detection enzyme. If the kit
is for use with initial PCR amplification then it could also
include the following components: (i) a pair of primers for PCR, at
least one primer having means permitting immobilization of said
primer; (ii) a polymerase which is preferably heat stable, for
example Taq1 polymerase; (iii) buffers for the PCR reaction; and
(iv) deoxynucleotides. Where an enzyme label is used to evaluate
PCR, the kit will advantageously contain a substrate for the enzyme
and other components of a detection system.
Mathematical Analysis Underlying Optimization of the Pyrophosphate
Sequencing Reaction
[0216] While not wishing to be bound by theory, it is believed that
optimization of reaction conditions can be performed using
assumptions underlying the following analyses.
[0217] Solid-phase pyrophosphate sequencing was initially developed
by combining a solid-phase technology and a sequencing-by-synthesis
technique utilizing bioluminescence (see e.g., Ronaghi, et al.,
1996. Real-time DNA sequencing using detection of pyrophosphate
release. Anal. Biochem. 242: 84-89). In the solid-phase
methodology, an immobilized, primed DNA strand is incubated with
DNA polymerase, ATP sulfurylase, and luciferase. By stepwise
nucleotide addition with intermediate washing, the event of
sequential polymerization can be followed. The signal-to-noise
ratio was increased by the use of .alpha.-thio dATP in the system.
This dATP analog is efficiently incorporated by DNA polymerase but
does not serve as a substrate for luciferase. This reduces
background bioluminescence and facilitates performance of the
sequencing reaction in real-time. In these early studies,
sequencing of a PCR product using streptavidin-coated magnetic
beads as a solid support was presented. However, it was found that
the loss of the beads during washing, which was performed between
each nucleotide and enzyme addition, limited the technique to short
sequences.
[0218] Currently, pyrophosphate sequencing methodologies have a
reasonably well-established history for ascertaining the DNA
sequence from many identical copies of a single DNA sequencing
template (see e.g., Ronaghi, et al., 1996. Real-Time DNA Sequencing
Using Detection of Pyrophosphate Release, Anal. Biochem. 242:
84-89; Nyren, et al., Method of Sequencing DNA, patent WO9813523A1
(issued Apr. 2, 1998; filed Sep. 26, 1997); Ronaghi, et al., 1998.
A Sequencing Method Based on Real-Time Pyrophosphate Science 281:
363-365 (1998). Pyrophosphate (PPi)-producing reactions can be
monitored by a very sensitive technique based on bioluminescence
(see e.g., Nyren, et al., 1996. pp. 466-496 (Proc. 9.sup.th Inter.
Symp. Biolumin. Chemilumin.). These bioluminometric assays rely
upon the detection of the PPi released in the different nucleic
acid-modifying reactions. In these assays, the PPi which is
generated is subsequently converted to ATP by ATP sulfurylase and
the ATP production is continuously monitored by luciferase. For
example, in polymerase-mediated reactions, the PPi is generated
when a nucleotide is incorporated into a growing nucleic acid chain
being synthesized by the polymerase. While generally, a DNA
polymerase is utilized to generate PPi during a pyrophosphate
sequencing reaction (see e.g., Ronaghi, et al., 1998. Doctoral
Dissertation, The Royal Institute of Technology, Dept. of
Biochemistry (Stockholm, Sweden)), it is also possible to use
reverse transcriptase (see e.g., Karamohamamed, et al., 1996. pp.
319-329 (Proc. 9.sup.th Inter. Symp. Biolumin. Chemilumin.) or RNA
polymerase (see e.g., Karamohamamed, et al., 1998. BioTechniques
24: 302-306) to follow the polymerization event.
[0219] For example, a bioluminometric primer extension assay has
been utilized to examine single nucleotide mismatches at the
3'-terminus (see e.g., Nyren, et al., 1997. Anal. Biochem. 244:
367-373). A phage promoter is typically attached onto at least one
of the arbitrary primers and, following amplification, a
transcriptional unit may be obtained which can then be subjected to
stepwise extension by RNA polymerase. The transcription-mediated
PPi-release can then be detected by a bioluminometric assay (e.g.,
ATP sulfurylase-luciferase). By using this strategy, it is likely
to be possible to sequence double-stranded DNA without any
additional specific sequencing primer. In a series of "run-off"
assays. the extension by T7 phage RNA polymerase has been examined
and was found to be rather slow (see e.g., Kwok, et al., 1990.
Nucl. Acids Res. 18: 999-1005). The substitution of an .alpha.-thio
nucleotide analogs for the subsequent, correct natural
deoxynucleotide after the 3'-mismatch termini, could decrease the
rate of polymerization by 5-fold to 13-fold. However, after
incorporation of a few bases, the rate of DNA synthesis is
comparable with the rate observed for a normal template/primer.
[0220] Single-base detection by this technique has been improved by
incorporation of apyrase to the system, which catalyzes NTP
hydrolysis and reduces the nucleotide concentration far below the
K.sub.m of DNA polymerase, effectively removing dNTP from a
preceding step before proceeding to addition of the subsequent
dNTP. The above-described technique provides a rapid and real-time
analysis for applications in the areas of mutation detection and
single-nucleotide polymorphism (SNP) analysis.
[0221] The pyrophosphate sequencing system uses reactions catalyzed
sequentially by several enzymes to monitor DNA synthesis. Enzyme
properties such as stability, specificity, sensitivity, K.sub.M and
k.sub.CAT are important for the optimal performance of the system.
In the pyrophosphate sequencing system, the activity of the
detection enzymes (i.e., sulfurylase and luciferase) generally
remain constant during the sequencing reaction, and are only very
slightly inhibited by high amounts of products (see e.g., Ronaghi,
et al., 1998. Doctoral Dissertation, The Royal Institute of
Technology, Dept. of Biochemistry (Stockholm, Sweden)). Sulfurylase
converts each PPi to ATP in approximately 2.0 seconds (see e.g.,
Nyren and Lundin, 1985. Anal. Biochem. 151: 504-509). The reported
reaction conditions for 1 pmol PPi in 0.2 ml buffer (5 nM) are 0.3
U/ml ATP sulfurylase (ATP:sulfate adenylyltransferase; Prod. No.
A8957; Sigma Chemical Co., St. Louis, Mo.) and 5 .mu.M APS (see
e.g., Ronaghi, et al., 1996. Real-Time DNA Sequencing Using
Detection of Pyrophosphate Release, Anal. Biochem. 242: 84-89). The
manufacturer's information (Sigma Chemical Co., St. Louis, Mo.) for
sulfurylase sports an activity of 5-20 units per mg protein (i.e.,
one unit will produce 1.0 .mu.mole of ATP from APS and PPi per
minute at pH 8.0 at 30 C). whereas the specific activity has been
reported elsewhere as 140 units per mg (see Karamohamed, et al.,
1999. Purification, and Luminometric Analysis of Recombinant
Saccharomyces cerevisiae MET3 Adenosine Triphosphate Sulfurylase
Expressed in Escherichia coli. Prot. Express. Purification 15:
381-388). Due to the fact that the reaction conditions utilized in
the practice of the present invention are similar to those reaction
conditions reported in the aforementioned reference, the
sulfurylase concentration within the assay was estimated as 4.6 nM.
The K.sub.M values for sulfurylase are [APS]=0.5 .mu.M and [PPi]=7
.mu.M. The generation of light by luciferase takes place in less
than 0.2 seconds. The most critical reactions are the DNA
polymerization and the degradation of nucleotides. The value of
constants characterizing the enzymes utilized in the pyrophosphate
sequencing methodology are listed below for reference:
TABLE-US-00001 Enzyme K.sub.M (.mu.M) k.sub.CAT (S.sup.-1) Klenow
0.18 (dTTP) 0.92 T.sub.7 DNA Polymerase 0.36 (dTTP) 0.52 ATP
Sulfurylase 0.56 (APS); 7.0 (PPi) 38 Firefly Luciferase 20 (ATP)
0.015 Apyrase 120 (ATP); 260 (ADP) 500 (ATP)
[0222] The enzymes involved in these four reactions compete for the
same substrates. Therefore, changes in substrate concentrations are
coupled. The initial reaction is the binding of a dNTP to a
polymerase/DNA complex for chain elongation. For this step to be
rapid, the nucleotide triphosphate concentration must be above the
K.sub.M of the DNA polymerase. If the concentration of the
nucleotide triphosphates is too high, however, lower fidelity of
the polymerase may be observed (see e.g., Cline, et al., 1996. PCR
fidelity of Pfu DNA polymerase and other thermostable DNA
polymerases. Nucl. Acids Res. 24: 3546-3551). A suitable range of
concentrations is established by the K.sub.M for the
misincorporation, which is usually much higher (see e.g., Capson,
et al., 1992. Kinetic characterization of the polymerase and
exonuclease activity of the gene 43 protein of bacteriophage T4.
Biochemistry 31: 10984-10994). Although a very high fidelity can be
achieved by using polymerases with inherent exonuclease activity,
their use also holds the disadvantage that primer degradation may
occur.
[0223] Although the exonuclease activity of the Klenow fragment of
DNA polymerase I (Klenow) is low, it has been demonstrated that the
3'-terminus of a primer may be degraded with longer incubations in
the absence of nucleotide triphosphates (see e.g., Ronaghi, et al.,
1998. Doctoral Dissertation, The Royal Institute of Technology,
Dept. of Biochemistry (Stockholm, Sweden)). Fidelity is maintained
without exonuclease activity because an induced-fit binding
mechanism in the polymerization step provides a very efficient
selectivity for the correct dNTP. Fidelities of 1.times.10.sup.5 to
1.times.10.sup.6 have been reported (see e.g., Wong, et al., 1991.
An induced-fit kinetic mechanism for DNA replication fidelity.
Biochemistry 30: 526-537). In pyrophosphate sequencing,
exonuclease-deficient (exo-) polymerases, such as exo-Klenow or
Sequenase.RTM., have been confirmed to have high fidelity.
[0224] Estimates for the spatial and temporal constraints on the
pyrophosphate sequencing methodology of the present invention have
been calculated, wherein the system possesses a 1 cm.sup.2 area
with height approximately 50 .mu.m, for a total volume of 5 .mu.l.
With respect to temporal constraints, the molecular species
participating in the cascade of reactions are initially defined,
wherein: [0225] N=the DNA attached to the surface [0226] PPi=the
pyrophosphate molecule released [0227] ATP=the ATP generated from
the pyrophosphate [0228] L=the light released by luciferase
[0229] It is further specified that N(0) is the DNA with no
nucleotides added, N(1) has 1 nucleotide added, N(2) has 2
nucleotides added, and so on. The pseudo-first-order rate constants
which relate the concentrations of molecular species are:
N(n).fwdarw.N(n+1)+PP.sub.i k.sub.N PPi.fwdarw.ATP k.sub.P
ATP.fwdarw.L k.sub.A
[0230] In addition, the diffusion constants D.sub.P for PPi and
D.sub.A for ATP must also be specified. These values may be
estimated from the following exemplar diffusion constants for
biomolecules in a dilute water solution (see Weisiger, 1997. Impact
of Extracellular and Intracellular Diffusion on Hepatic Uptake
Kinetics). TABLE-US-00002 Molecule D/10.sup.-5 cm.sup.2/sec Method
Original Reference Albumin 0.066 lag time 1 Albumin 0.088 light
scattering 2 Water 1.940 NMR 3
wherein, Original Reference 1 is: Longsworth, 1954. Temperature
dependence of diffusion in aqueous solutions, J. Phys. Chem. 58:
770-773; Original Reference 2 is: Gaigalas, et al., 1992. Diffusion
of bovine serum albumin in aqueous solutions, J. Phys. Chem. 96:
2355-2359; and Original Reference 3 is: Cheng, 1993. Quantitation
of non-Einstein diffusion behavior of water in biological tissues
by proton NMR diffusion imaging: Synthetic image calculations,
Magnet. Reson. Imaging 11: 569-583.
[0231] In order to estimate the diffusion constant of PPi, the
following exemplar values may be utilized (see CRC Handbook of
Chemistry and Physics, 1983. (W. E. Weast. Ed.) CRC Press, Inc.,
Boca Raton, Fla.): TABLE-US-00003 Molecule D/10.sup.-5 cm.sup.2/sec
Molecular Weight/amu sucrose 0.5226 342.30 mannitol 0.682 182.18
penta-erythritol 0.761 136.15 glycolamide 1.142 N/A glycine 1.064
75.07
[0232] The molecular weight of PPi is 174 amu. Based upon the
aforementioned exemplar values, a diffusion constant of
approximately 0.7.times.10.sup.-5 cm.sup.2/sec for PPi is
expected.
[0233] Enzymes catalyzing the three pyrophosphate sequencing
reactions are thought to approximate Michaelis-Menten kinetics (see
e.g. Stryer, 1988. Biochemistry, W. H. Freeman and Company, New
York), which may be described: K.sub.M=[E][S]/[ES],
velocity=V.sub.max[S]/(K.sub.M+[S]), V.sub.max=k.sub.CAT[E.sub.T]
where [S] is the concentration of substrate, [E] is the
concentration of free enzyme, [ES] is the concentration of the
enzyme-substrate complex, and [E.sub.T] is the total concentration
of enzyme=[E]+[ES].
[0234] It is preferable that the reaction times are at least as
fast as the solution-phase pyrophosphate-based sequencing described
in the literature. That rate that a substrate is converted into
product is -d[S]/dt=k.sub.CAT[E.sub.T][S]/(K.sub.M+[S])
[0235] The effective concentration of substrate may be estimated
from the size of a replicated DNA molecule, at most (10
.mu.m).sup.3 and the number of copies (approximately 10,000),
yielding a concentration of approximately 17 nM. This is this is
smaller than the K.sub.M for the enzymes described previously, and
therefore the rate can be estimated to be
-d[S]/dt=(k.sub.CAT/K.sub.M)[E.sub.T][S].
[0236] Thus, with pseudo first-order kinetics, the rate constant
for disappearance of substrate depends on k.sub.CAT and K.sub.M,
which are constants for a given enzyme, and [E.sub.T]. Using the
same enzyme concentrations reported in the literature will
therefore produce similar rates.
[0237] The first step in the pyrophosphate sequencing reaction
(i.e., incorporation of a new nucleotide and release of PPi) will
now be examined in detail. The preferred reaction conditions are: 1
pmol DNA, 3 pmol polymerase, 40 pmol dNTP in 0.2 ml buffer. Under
the aforementioned, preferred reaction conditions, the K.sub.M for
nucleotide incorporation for the Klenow fragment of DNA polymerase
I is 0.2 .mu.M and for Sequenase 2.0.TM. (US Biochemicals,
Cleveland, Ohio) is 0.4 .mu.M, and complete incorporation of 1 base
is less than 0.2 sec (see e.g., Ronaghi, et al., 1996. Real-Time
DNA Sequencing Using Detection of Pyrophosphate Release, Anal.
Biochem. 242: 84-89) with a polymerase concentration of 15 nM.
[0238] In a 5 .mu.l reaction volume, there are a total of 10,000
anchor primers with 10,000 sequencing primer sites each, or
1.times.10.sup.8 total extension sites=0.17 fmol. Results which
have been previously published in the literature suggest that
polymerase should be present at 3-times abundance, or 0.5 fmol,
within the reaction mixture. The final concentration of polymerase
is then 0.1 nM. It should be noted that these reaction conditions
are readily obtained in the practice of tic present invention.
[0239] As previously stated, the time required for the nucleotide
addition reaction is no greater than 0.2 sec per nucleotide. Hence,
if tie reaction is allowed to proceed for a total of T seconds,
then nucleotide addition should be sufficiently rapid that
stretches of up to (T/0.2) identical nucleotides should be
completely filled-in by the action of the polymerase. As discussed
previously, the rate-limiting step of the pyrophosphate sequencing
reaction is the sulfurylase reaction, which requires a total of
approximately 2 sec to convert one PPi to ATP. Accordingly, a total
reaction time which allows completion of the sulfurylase reaction,
should be sufficient to allow the polymerase to "fill-in" stretches
of up to 10 identical nucleotides. In random DNA species, regions
of 10 or more identical nucleotides have been demonstrated to occur
with a per-nucleotide probability of approximately 4.sup.-10, which
is approximately 1.times.10.sup.-6. In the 10,000 sequences which
are extended from anchor primers in a preferred embodiment of the
present invention, each of which will be extended at least 30
nucleotides and preferably 100 nucleotides, it is expected that
approximately one run of 10 identical nucleotides will be present.
Thus, it may be concluded that runs of identical nucleotides should
not pose a difficulty in the practice of the present invention.
[0240] The overall size of the resulting DNA molecule is,
preferably, smaller than the size of the anchoring pads (i.e., 10
.mu.m) and must be smaller than the distance between the individual
anchoring pads (i.e., 100 .mu.m). The radius of gyration of a
single-stranded DNA concatamer with N total nucleotides may be
mathematically-estimated by the following equation: radius=b
(N/N.sub.0).sup.0.6, where b is the persistence length and N.sub.0
is the number of nucleotides per persistence length; the exponent
0.6 is characteristic of a self-avoiding walk (see e.g., Doi, 1986.
The Theory of Polymer Dynamics (Clarendon Press, New York); Flory,
1953. Principles of Polymer Chemistry (Cornell University Press,
New York)). Using single-stranded DNA as an example, b is 4 nm and
N.sub.0 is 13.6 nucleotides. (see e.g., Grosberg, 1994. Statistical
Physics of Macromolecules (AIP Press, New York)). Using 10,000
copies of a 100-mer, N=1.times.10.sup.6 and the radius of gyration
is 3.3 .mu.m.
[0241] The diffusion of PPi will now be discussed in detail. In the
reaction conditions utilized in the present invention, [PP.sub.i]
is approximately 0.17 fmol in 5 .mu.l, or 0.03 nM, and
[sulfurylase] is 4.6 nM as described previously. In the first 2 sec
of the reaction, about 7% (0.002 nM) of PPi is consumed by
sulfurylase, using GEPASI simulation software (see Mendes, P.
(1993) GEPASI: a software package for modeling the dynamics, steady
states and control of biochemical and other systems. Comput. Appl.
Biosci. 9, 563-571.). The parameters used in simulation were
K.sub.M(PPi)=7 .mu.M, k.sub.CAT--38 s.sup.-1, and [sulfurylase]=4.6
nM. Therefore, it may be concluded that at least 93% of PPi
molecules may diffuse away before being converted to ATP (during
the 2 sec reaction time.
[0242] The mean time for each PPi to react is 1/k.sub.P=2 seconds.
The mean square distance it diffuses in each direction is
approximately 2D.sub.P/k.sub.P, or 2.8.times.10.sup.3 .mu.m.sup.2.
The RMS distance in each direction is 53 .mu.m. This value
indicates that each of the individual anchor primers must be more
than 50 .mu.m apart, or PPi which is released from one anchor could
diffuse to the next, and be detected.
[0243] Another method which may be used to explain the
aforementioned phenomenon is to estimate the amount of PPi over a
first anchor pad that was generated at said first anchor pad
relative to the amount of PPi that was generated at a second anchor
pad and subsequently diffused over to the location of said first
anchor pad. When these two quantities approach each other in
magnitude, it becomes difficult to distinguish the "true" signal
from that of the background. This may be mathematically-described
by defining a as the radius of an anchor pad and 1/b.sup.2as the
density of an anchor pad. Based upon previously published data, a
is approximately equal to 10 .mu.m and b is approximately equal to
100 .mu.m. The amount of PPi which is present over said first
anchor pad may be described by:
exp(-k.sub.Pt)[1-exp(-a.sup.2/2D.sub.Pt)] and the amount of PPi
present over the second anchor pads may be mathematically
approximated by:
[0244]
(1/3)exp(-k.sub.Pt)[pa.sup.2/b.sup.2]exp(-b.sup.2/2D.sub.Pt). The
prefactor 1/3 assumes that 1/4 of the DNA sequences will
incorporate 1 nucleotide, 1/4 of these will then incorporate a
second nucleotide, etc., and thus the sum of the series is 1/3. The
amounts of PPi over the first and second anchor pads become similar
in magnitude when 2D.sub.Pt is approximately equal to b.sup.2, thus
indicating that the RMS distance a molecule diffuses is equal to
the distance between adjacent anchor pads. In accord, based upon
the assay conditions utilized in the practice of the present
invention, the anchor pads must be placed no closer than
approximately 50 .mu.m apart, and preferable are at least 3-times
further apart (i.e., 150 .mu.m).
[0245] Although the aforementioned findings set a limit on the
surface density of anchor pads, it is possible to decrease the
distance requirements, while concomitantly increasing the overall
surface density of the anchor pads, by the use of a number of
different approaches. One approach is to detect only the early
light, although this has the disadvantage of losing signal,
particularly from DNA sequences which possess a number of
contiguous, identical nucleotides.
[0246] A second approach to decrease the distance between anchor
pads is to increase the concentration of sulfurylase in the
reaction mixture. The reaction rate k.sub.P is directly
proportional to the sulfurylase concentration, and the diffusion
distance scales as k.sub.P.sup.-1/2. Therefore, if the sulfurylase
enzyme concentration is increased by a factor of 4-times, the
distance between individual anchor pads may be concomitantly
reduced by a factor of 2-times.
[0247] A third approach is to increase the effective concentration
of sulfurylase (which will also work for other enzymes described
herein) by binding the enzyme to the surface of (he anchor pads.
The anchor pad can be approximated as one wall of a cubic surface
enclosing a sequencing reaction center. Assuming a 10
.mu.m.times.10 .mu.m surface for the pad, the number of molecules
bound to the pad to produce a concentration of a 1 .mu.M is
approximately 600,000 molecules.
[0248] The sulfurylase concentration in the assay is estimated as 5
nM. The number of bound molecules to reach this effective
concentration is about 3000 molecules. Thus, by binding more enzyme
molecules, a greater effective concentration will be attained. For
example, 10,000 molecules could be bound per anchor pad.
[0249] As previously estimated, each sulfurylase molecule occupies
a total area of 65 nm.sup.2 on a surface. Accordingly, anchoring a
total of 10,000 sulfurylase enzyme molecules on a surface (i.e., so
as to equal the 10,000 PPi released) would require 1.7 .mu.m.sup.2.
This value is only approximately 2% of the available surface area
on a 10 .mu.m.times.10 .mu.m anchor pad. Hence, the concentration
of the enzyme may be readily increased to a much higher value.
[0250] A fourth approach to allow a decrease in the distance
between individual anchor pads, is to utilize one or more agents to
increase the viscosity of the aqueous-based, pyrophosphate
sequencing reagents (e.g., glycerol, polyethylene glycol (PEG), and
the like) so as to markedly increase the time it takes for the PPi
to diffuse. However, these agents will also concomitantly increase
the diffusion time for other non-immobilized components within the
sequencing reaction, thus slowing the overall reaction kinetics.
Additionally, the use of these agents may also function to
chemically-interfere with the sequencing reaction itself.
[0251] A fifth, and preferred, methodology to allow a decrease in
the distance between individual anchor pads, is to conduct the
pyrophosphate sequencing reaction in a spatial-geometry which
physically-prevents the released PPi from diffusing laterally. For
example, uniform cavities or microwells, such as those generated by
acid-etching the termini of optical fiber bundles, may be utilized
to prevent such lateral diffusion of PPi (see Michael, et al.,
1998. Randomly Ordered Addressable High-Density Optical Sensor
Arrays, Anal. Chem. 70: 1242-1248). In this embodiment, the
important variable involves the total diffusion time for the PPi to
exit a cavity of height h, wherein h is the depth of the etched
cavity. This diffusion time may be calculated utilizing the
equation: 2D.sub.Pt=h.sup.2. By use of the preferred pyrophosphate
sequencing reaction conditions of the present invention in the
aforementioned calculations, it may be demonstrated that a cavity
50 .mu.m in depth would be required for the sequencing reaction to
proceed to completion before complete diffusion of the PPi from
said cavity. Moreover, this type of geometry has the additional
advantage of concomitantly reducing background signal from the PPi
released from adjacent anchor pads.
[0252] Additionally, to prevent background generated by diffusion
of PPi from one pad to another, the region of substrate between the
pads can be coated with immobilized phosphatase.
[0253] Subsequently, once ATP has been formed by use of the
preferred reaction conditions of the present invention, the
reaction time, 1/k.sub.A, has been shown to be 0.2 seconds. Because
this reaction time is much lower than the time which the PPi is
free to diffuse, it does not significantly alter any of the
aforementioned conclusions regarding the assay geometry and
conditions utilized in the present invention.
[0254] In order to mitigate the generation of background light, it
is preferable to "localize" (e.g., by anchoring or binding) the
luciferase in the region of the DNA sequencing templates. It is
most preferable to localize the luciferase to a region that is
delineated by the distance a PPi molecule can diffuse before it
forms ATP. Methods for binding luciferase to a solid support matrix
are well-known in the literature (see e.g., Wang, et al, 1997.
Specific Immobilization of Firefly Luciferase through a Biotin
Carboxyl Carrier Protein Domain, Analytical Biochem. 246: 133-139).
Thus, for a 2 second diffusion time, the luciferase is anchored
within a 50 .mu.m distance of the DNA strand. It should be noted,
however, that it would be preferable to decrease the diffusion time
and thus to further limit the surface area which is required for
luciferase binding.
[0255] Additionally, to prevent background generated by diffusion
of ATP from one pad to another, the region of substrate between the
pads can be coated with immobilized ATPase, especially one that
hydrolyzes ATP to ADP, e.g. alkaline phosphatase.
[0256] In order to determine the concentration of luciferase which
it is necessary to bind, previously published conditions were
utilized in which luciferase is used at a concentration which gives
a response of 200 mV for 0.1 .mu.m ATP (see Ronaghi, et al., 1996.
Real-Time DNA Sequencing Using Detection of Pyrphosphate Release,
Analytical Biochem. 242: 84-89). More specifically, it is known
from the literature that, in a 0.2 ml reaction volume, 2 ng of
luciferase gives a response of 10 mV for 0.1 .mu.M ATP (see
Karamohamed and Nyren, 1999. Real-Time Detection and Quantification
of Adenosine Triphosphate Sulfurylase Activity by a Bioluminometric
Approach, Analytical Biochem. 271: 81-85). Accordingly, a
concentration of 20 ng of luciferase within a 0.2 ml total reaction
volume would be required to reproduce these previously-published
literature conditions. In the volume of a 10 .mu.m cube around each
of the individual anchor pads of the present invention, a
luciferase concentration of 1.times.10.sup.-16 grams would be
required, and based upon the 71 kDa molecular weight of luciferase,
this concentration would be equivalent to approximately 1000
luciferase molecules. As previously stated, the surface area of
luciferase has been computed at 50 nm.sup.2. Thus, assuming the
luciferase molecules were biotinylated and bound to the anchor pad,
1000 molecules would occupy a total area of 0.05 .mu.m.sup.2. From
these calculations it becomes readily apparent that a plethora of
luciferase molecules may be bound to the anchor pad, as the area of
each anchor pad area is 100 .mu.m.sup.2.
[0257] Again, based upon previously published results in the
literature, each nucleotide takes approximately 3 seconds to
sequence (i.e., 0.2 second to add a nucleotide; 2 seconds to make
ATP; 0.2 seconds to get bioluminescence). Accordingly, a cycle time
of approximately 60 seconds per nucleotide is reasonable, requiring
approximately 30 minutes per experiment to generate 30 nucleotides
of information per sequencing template.
[0258] In an alternative embodiment to the aforementioned
sequencing methodology (i.e.,
polymerase.fwdarw.PPi.fwdarw.sulfurylase.fwdarw.ATP.fwdarw.luciferase.fwd-
arw.light), a polymerase may be developed (e.g., through the use of
protein fusion and the like) which possesses the ability to
generate light when it incorporates a nucleotide into a growing DNA
chain. In yet another alternative embodiment, a sensor may be
developed which directly measures the production of PPi in the
sequencing reaction. As the production of PPi changes the electric
potential of the surrounding buffer, this change could be measured
and calibrated to quantify the concentration of PPi produced.
[0259] As previously discussed, the polymerase-mediated
incorporation of dNTPs into the nucleotide sequence in the
pyrophosphate sequencing reaction causes the release of a photon
(i.e., light). The photons generated by the pyrophosphate
sequencing reaction may subsequently be "captured" and quantified
by a variety of methodologies including, but not limited to: a
photomultiplier tube, CCD, absorbance photometer, a luminometer,
and the like.
[0260] The photons generated by the pyrophosphate sequencing
reaction are captured by the CCD. The efficiency of light capture
increases if they pass through a focusing device (e.g., an optical
lens or optical fiber) and are focused upon a CCD element. The
fraction of these photons which are captured may be estimated by
the following calculations. First, it is assumed that the lens that
focuses the emitted photons is at a distance r from the surface of
the solid surface (i.e., DNA chip or etched fiber optic well),
where r=1 cm and that the photons must pass through a region of
diameter b (area=.pi.b.sup.2/4) so as to be focused upon the array
element, where b=100 .mu.m. (This produces an optical system with
numerical aperture of approximately 0.01 in air.) It should also be
noted that the emitted photons should escape equally in all
directions. At distance r, the photons are dispersed over an area
of which is equal to 4.pi.r.sup.2. Thus, the fraction of photons
which pass through the lens is described by:
(1/2)[1-(1+b.sup.2/4r.sup.2).sup.-1/2]. When the value of r is much
larger than that of b, the fraction which pass through the lens may
then be described by: b.sup.2/16r.sup.2. For the aforementioned
values of r and b, this fraction of photons is 6.times.10.sup.-6.
Note that the fraction of captured photons increases as b increases
or r decreases (i.e. as the numerical aperture of the imaging
system increases). Use of FORA in which the microwells are etched
into the termini of optical fibers, which then also serve to focus
the light onto a CCD, greatly increases the numerical aperture from
the example given above, with the numerical aperture of many fiber
optics being in the range of 0.7. For each nucleotide addition, it
is expected that approximately 10,000 PPi molecules will be
generated and, if all are converted by sulfurylase and luciferase,
these PPi will result in the emission of approximately
1.times.10.sup.4 photons. In order to maximize their subsequent
"capture" and quantitation when utilizing a planar array (e.g., a
DNA chip), it is preferable to collect the photons immediately at
the planar solid support (e.g., the cover slip). This may be
accomplished by either: (i) utilizing optical immersion oil between
the cover slip and a traditional optical lens or optical fiber
bundle or, preferably, (ii) incorporating optical fibers directly
into the cover slip itself. Performing the previously described
calculations (where in this case, b=100 .mu.m and r=50 .mu.m), the
fraction collected is found to be 0.15, which equates to the
capture of approximately 1.times.10.sup.3 photons. This value would
be sufficient to provide an adequate signal.
[0261] The following examples are meant to illustrate, not limit,
the invention.
Example 1
Construction of Anchor Primers Linked to a Cavitated Terminus Fiber
Optic Array
[0262] The termini of a thin wafer fiber optic array are cavitated
by inserting the termini into acid as described by Healey et al.,
Anal Chem. 69: 2213-2216 (1997).
[0263] A thin layer of a photoactivatable biotin analog is dried
onto the cavitated surface as described in Hengsakul and Cass
(Bioconjugate Chem. 7: 249-254, 1996) and exposed to white light
through a mask to create defined pads, or areas of active biotin.
Next, avidin is added and allowed to bind to the biotin.
Biotinylated oligonucleotides are then added. The avidin has free
biotin binding sites that can anchor biotinylated oligonucleotides
through a biotin-avidin-biotin link.
[0264] The pads are approximately 10 .mu.m on a side with a 100
.mu.m spacing. Oligonucleotides are added so that approximately 37%
of the pads include one anchored primer. On a 1 cm.sup.2 surface
are deposited 10,000 pads, yielding approximately 3700 pads with a
single anchor primer.
Example 2
Annealing and Amplification of Members of a Circular Nucleic Acid
Library
[0265] A library of open circle library templates is prepared from
a population of nucleic acids suspected of containing a single
nucleotide polymorphism on a 70 bp Sau3A1-MspI fragment. The
templates include adapters that are complementary to the anchor
primer, a region complementary to a sequencing primer, and an
insert sequence that is to be characterized. The library is
generated using Sau3A1 and MspI to digest the genomic DNA. Inserts
approximately 65-75 nucleotides are selected and ligated to adapter
oligonucleotides 12 nucleotides in length. The adapter
oligonucleotides have sequences complementary to sequences to an
anchor primers linked to a substrate surface as described in
Example 1.
[0266] The library is annealed to the array of anchor primers. A
DNA polymerase is added, along with dNTPs, and rolling circle
replication is used to extend the anchor primer. The result is a
single DNA strand, still anchored to the solid support, that is a
concatenation of multiple copies of the circular template. 10,000
or more copies of circular templates in the hundred nucleotide size
range.
Example 3
Sequence Analysis of Nucleic Acid Linked to the Terminus of a Fiber
Optic Substrate
[0267] The fiber optic array wafer containing amplified nucleic
acids as described in Example 2 is placed in a perfusion chamber
and attached to a bundle of fiber optic arrays, which are
themselves linked to a 16 million pixel CCD camera. A sequencing
primer is delivered into the perfusion chamber and allowed to
anneal to the amplified sequences. Then sulfurylase, apyrase, and
luciferase are attached to the cavitated substrate using
biotin-avidin.
[0268] The sequencing primer primes DNA synthesis extending into
the insert suspected of having a polymorphism, as shown in FIG. 1.
The sequencing primer is first extended by delivering into the
perfusion chamber, in succession, a wash solution, a DNA
polymerase, and one of dTTP, dGTP, dCTP, or .alpha. thio dATP (a
dATP analog). The sulfurylase, luciferase, and apyrase, attached to
the termini convert any PPi liberated as part of the sequencing
reaction to detectable light. The apyrase present degrades any
unreacted dNTP. Light is typically allowed to collect for 3 seconds
(although 1-100, e.g., 2-10 seconds is also suitable) by a CCD
camera linked to the fiber imaging bundle, after which additional
wash solution is added to the perfusion chamber to remove excess
nucleotides and byproducts. The next nucleotide is then added,
along with polymerase, thereby repeating the cycle.
[0269] During the wash the collected light image is transferred
from the CCD camera to a computer. Light emission is analyzed by
the computer and used to determine whether the corresponding dNTP
has been incorporated into the extended sequence primer. Addition
of dNTPs and pyrophosphate sequencing reagents is repeated until
the sequence of the insert region containing the suspected
polymorphism is obtained.
Example 4
Sequence Analysis of a Tandem Repeat Template Generated Using
Rolling Circle Amplification
[0270] A primer having the sequence 5'-gAC CTC ACA CgA Tgg CTg CAg
CTT-3' (SEQ ID NO:2) was annealed to a 88 nucleotide template
molecule having the sequence 5'-TCg TgT gAg gTC TCA gCA TCT TAT gTA
TAT TTA CTT CTA TTC TCA gTT gCC TAA gCT gCA gCC A-3' (SEQ ID NO:1).
Annealing of the template to the primer resulted in juxtaposition
of the 5' and 3' ends of the template molecule. The annealed
template was exposed to ligase, which resulted in ligation of the
5' and 3' ends of the template to generate a circular molecule.
[0271] The annealed primer was extended using Klenow fragment and
nucleotides in rolling circle amplification for 12 hours at
37.degree. C. The product was purified using the SPRI technique
(Seradyn, Indianapolis, Ind.). Rolling circle amplification
resulted in formation of tandem repeats of a sequence complementary
to the circular template sequence.
[0272] The tandem repeat product in the extended sequence was
identified by annealing a sequencing primer having the sequence
5'-AAgCTgCAgCCATCgTgTgAgg-3' (SEQ ID NO:8) and subjecting the
annealed primer to 40 alternating cycles of 95.degree. C., 1
minute, 20 seconds, 60.degree. C. using ET terminator chemistry
(Amersham-Pharmacia) in the presence of 1M betaine.
[0273] The sequencing product was then diluted to 1/5 volume and
purified on a G-50 Sephadex column prior to injection into a
MegaBACE sequencing system with linear polyacrylamide
(Amersham-Pharmacia).
[0274] An electropherogram of the sequencing analysis is shown in
FIG. 5. The tracing demonstrates that multiple copies of the 88 bp
circular template molecule are generated tandemly, and that these
copies can be detected in a DNA sequencing reaction.
Example 5
FORA Preparation
[0275] DNA beads: Deoxyoligonucleotide--ggggAATTCAAAATTTggC (SEQ ID
NO:9) were annealed to capture probes, which were biotinylated at
the 5' end, and then immobilized on either Dynal M-280 (Dynal) or
MPG beads (CPG) (bead concentration was 1 mg/ml). The
immobilization was carried out by incubating the beads, with a
fixed amount of oligonucleotide for 30 minutes. Different loadings
of oligonucleotide were obtained by changing amount of
oligonucleotide used during incubation. After incubation, the beads
were washed in respective volumes of TE buffer and resuspened in
same volumes of TE.
[0276] Enzyme beads: A mixture of 1:1 (vol/vol) of sulfurylase (1
mg/mL) and luciferase (3 mg/mL) with BBCP domains on their
N-termini were incubated with equal volume of Dynal M-280 (Dynal)
(concentration: 10 mg/mL) for one hour at 4.degree. C. After an
hour of incubation the beads were washed with assay buffer (25 mM
Tricine, 5 mM MgOAc and 1 mg/mL BSA) four times and then
resuspended in same volume of assay buffer.
[0277] FORA Preparation: The DNA beads were diluted 10 times to a
final concentration of 0.1 mg/mL before use. The enzyme beads were
used at 10 mg/mL concentration. The FORA was placed in jig which
has 10 spots created by O-rings (3 mm in diameter). 5 uL of DNA
beads were delivered, in 9 spots. The first spot on the inlet was a
control spot, with no DNA, to detect any background in the
reagents. The jig was placed in a centrifuge and spun at 2000 rpm
for five minutes. The centrifugal force, forces the beads to the
bottom of the wells (approximately 5-10 beads/well) The jig is
removed from the centrifuge and 5 uL of SL beads are added and the
jig is placed in the centrifuged and the spun at 2000 rpm for five
minutes. The process is repeated with 5 uL of SL beads. The FORA is
removed from the jig, placed in a falcon tube containing assay
buffer and washed by a gentle rocking motion three to four times.
The FORA thus prepared is ready for sequence analysis by
pyrophosphate sequencing.
Example 6
Sequence Analysis of Nucleic Acid Linked to the Terminus of a Fiber
Optic Substrate
[0278] Reagents: Reagents used for sequence analysis and as
controls were the four nucleotides and 0.1 .mu.M Pyrophosphate
(PPi) were made in substrate solution, where substrate refers to a
mixture of 300 .mu.M Luciferin and 4 .mu.M adenosine
5'-phosphosulfate, APS, which are the substrates for the cascade of
reactions involving PPi, Luciferase and Sulfurylase. The substrate
was made in assay buffer. The concentration of PPi used to test the
enzymes and determine the background levels of reagents passing
through the chamber was 0.1 .mu.M. The concentration of the
nucleotides, dTTP, dGTP, dCTP was 6.5 .mu.M and that of .alpha.dATP
was 50 .mu.M. Each of the nucleotides was mixed with DNA
polymerase, Klenow at a concentration of 100 U/mL.
[0279] The FORA was placed in the flow chamber of the embodied
instrument, and the flow chamber was attached to the faceplate of
the CCD camera. The FORA was washed by flowing substrate (3 ml per
min, 2 min) through the chamber. Subsequently, a sequence of
reagents was flown through the chamber by the pump connected to an
actuator, which was programmed to switch positions, which had tubes
inserted in the different reagents. The camera was set up in a fast
acquisition mode, with exposure time=2.5 s.
[0280] The signal output from the pad is the average of counts on
all the pixels within the pad. The frame number is equivalent of
the time passed during the experiment. The graph indicates the flow
of the different reagents.
OTHER EMBODIMENTS
[0281] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
9 1 64 DNA Homo sapiens 1 tcgtgtgagg tctcagcatc ttatgtatat
ttacttctat tctcagttgc ctaagctgca 60 gcca 64 2 24 DNA Artificial
Sequence Description of Artificial Sequence anchor primer 2
gacctcacac gatggctgca gctt 24 3 24 DNA Artificial Sequence
Description of Artificial Sequence anchor primer 3 gacctcacac
gatggctgca gctt 24 4 64 DNA Artificial Sequence Description of
Artificial Sequence SNP probe 4 tttatatgta ttctacgact ctggagtgtg
ctaccgacgt cgaatccgtt gactcttatc 60 ttca 64 5 34 DNA Artificial
Sequence Description of Artificial Sequence SNP region of gene 5
ctagctcgta catataaatg aagataagat cctg 34 6 30 DNA Artificial
Sequence Description of Artificial Sequence anchor primer 6
gacctcacac gagtagcatg gctgcagctt 30 7 64 DNA Homo sapiens 7
tcgtgtgagg tctcagcatc ttatgtatat ttacttctat tctcagttgc ctaagctgca
60 gcca 64 8 22 DNA Artificial Sequence Description of Artificial
Sequence sequencing primer 8 aagctgcagc catcgtgtga gg 22 9 19 DNA
Homo sapiens 9 ggggaattca aaatttggc 19
* * * * *