U.S. patent application number 11/235796 was filed with the patent office on 2006-02-09 for methods to increase nucleotide signals by raman scattering.
This patent application is currently assigned to INTEL CORPORATION. Invention is credited to Andrew A. Berlin, Selena Chan, Tae-Woong Koo, Xing Su, Narayan Sundararajan, Mineo Yamakawa.
Application Number | 20060029969 11/235796 |
Document ID | / |
Family ID | 28039551 |
Filed Date | 2006-02-09 |
United States Patent
Application |
20060029969 |
Kind Code |
A1 |
Su; Xing ; et al. |
February 9, 2006 |
Methods to increase nucleotide signals by Raman scattering
Abstract
The methods and apparatus disclosed herein concern nucleic acid
sequencing by enhanced Raman spectroscopy. In certain embodiments
of the invention, nucleotides are covalently attached to Raman
labels before incorporation into a nucleic acid 13. Exonuclease 15
treatment of the labeled nucleic acid 13 results in the release of
labeled nucleotides 16, 130, which are detected by Raman
spectroscopy. In alternative embodiments of the invention,
nucleotides 16, 130 released from a nucleic acid 13 by exonuclease
15 treatment are covalently cross-linked to silver or gold
nanoparticles 140 and detected by surface enhanced Raman
spectroscopy (SERS), surface enhanced resonance Raman spectroscopy
(SERFS) and/or coherent anti-Stokes Raman spectroscopy (CARS).
Other embodiments of the invention concern apparatus 10, 100, 210
for nucleic acid sequencing.
Inventors: |
Su; Xing; (Cupertino,
CA) ; Chan; Selena; (Sunnyvale, CA) ; Berlin;
Andrew A.; (San Jose, CA) ; Koo; Tae-Woong;
(South San Francisco, CA) ; Sundararajan; Narayan;
(San Francisco, CA) ; Yamakawa; Mineo; (Campbell,
CA) |
Correspondence
Address: |
Lisa A. Haile, J. D.;DLA PIPER RUDNICK GRAY CARY US LLP
Attorneys for INTEL CORPORATION
4365 Executive Drive, Suite 1100
San Diego
CA
92121-2133
US
|
Assignee: |
INTEL CORPORATION
|
Family ID: |
28039551 |
Appl. No.: |
11/235796 |
Filed: |
September 26, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10099287 |
Mar 14, 2002 |
6972173 |
|
|
11235796 |
Sep 26, 2005 |
|
|
|
Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 2563/155 20130101;
C12Q 2565/632 20130101; C12Q 2521/319 20130101; C12Q 2521/319
20130101; C12Q 2563/155 20130101; C12Q 1/6869 20130101; C12Q 1/6872
20130101; C12Q 1/6872 20130101; C12Q 1/6825 20130101; C12Q 1/6816
20130101; C12Q 1/6869 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-23. (canceled)
24. An apparatus comprising: a) a reaction chamber; b) a first
channel in fluid communication with said reaction chamber; c) a
second channel in fluid communication with said first channel; d) a
flow-through cell in fluid communication with said first and second
channels; and e) a detection unit operably coupled to said
flow-through cell.
25. The apparatus of claim 24, wherein said detection unit
comprises a Raman detector.
26. The apparatus of claim 25, wherein said detection unit
comprises a laser and a CCD camera.
27. The apparatus of claim 24, said first channel to contain
nucleotides and said second channel to contain nanoparticles.
28. The apparatus of claim 27, wherein said nucleotides are
covalently attached to said nanoparticles.
29. The apparatus of claim 28, wherein said nucleotides become
covalently attached to said nanoparticles within said channels.
30. The apparatus of claim 28, wherein covalent attachment of said
nucleotides to said nanoparticles provides an enhanced Raman
signal.
Description
FIELD OF THE INVENTION
[0001] The present methods and apparatus relate to the fields of
molecular biology and genomics. More particularly, the methods and
apparatus concern nucleic acid sequencing.
BACKGROUND
[0002] Genetic information is stored in the form of very long
molecules of deoxyribonucleic acid (DNA), organized into
chromosomes. The human genome contains approximately three billion
bases of DNA sequence. This DNA sequence information determines
multiple characteristics of each individual. Many common diseases
are based at least in part on variations in DNA sequence.
[0003] Determination of the entire sequence of the human genome has
provided a foundation for identifying the genetic basis of such
diseases. However, a great deal of work remains to be done to
identify the genetic variations associated with each disease. That
would require DNA sequencing of portions of chromosomes in
individuals or families exhibiting each such disease, in order to
identify specific changes in DNA sequence that promote the disease.
Ribonucleic acid (RNA), an intermediary molecule in processing
genetic information, may also be sequenced to identify the genetic
bases of various diseases.
[0004] Existing methods for nucleic acid sequencing, based on
detection of fluorescently labeled nucleic acids that have been
separated by size, are limited by the length of the nucleic acid
that can be sequenced. Typically, only 500 to 1,000 bases of
nucleic acid sequence can be determined at one time. This is much
shorter than the length of the functional unit of DNA, referred to
as a gene, which can be tens or even hundreds of thousands of bases
in length. Using current methods, determination of a complete gene
sequence requires that many copies of the gene be produced, cut
into overlapping fragments and sequenced, after which the
overlapping DNA sequences may be assembled into the complete gene.
This process is laborious, expensive, inefficient and
time-consuming.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the disclosed embodiments of the invention. The
embodiments of the invention may be better understood by reference
to one or more of these drawings in combination with the detailed
description of specific embodiments of the invention presented
herein.
[0006] FIG. 1 illustrates an exemplary apparatus 10 (not to scale)
and method for nucleic acid 13 sequencing, using nucleotides 16
covalently attached to Raman labels.
[0007] FIG. 2 illustrates an exemplary apparatus 100 (not to scale)
and method for nucleic acid 13 sequencing in which the released
nucleotides 130 are covalently attached to nanoparticles 140 prior
to detection by surface enhance Raman spectroscopy (SERS) 180.
[0008] FIG. 3 illustrates another exemplary apparatus 210 (not to
scale) for nucleic acid 13 sequencing.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0009] The disclosed methods and apparatus are of use for the
rapid, automated sequencing of nucleic acids 13. In particular
embodiments of the invention, the methods and apparatus 10, 100,
210 are suitable for obtaining the sequences of very long nucleic
acid molecules 13 of greater than 1,000, greater than 2,000,
greater than 5,000, greater than 10,000 greater than 20,000,
greater than 50,000, greater than 100,000 or even more bases in
length. Advantages over prior art methods include the ability to
read long nucleic acid 13 sequences in a single sequencing run,
greater speed of obtaining sequence data, decreased cost of
sequencing and greater efficiency in operator time required per
unit of sequence data.
[0010] In various embodiments of the invention, sequence
information may be obtained during the course of a single
sequencing run, using a single nucleic acid molecule 13. In other
embodiments of the invention, multiple copies of a nucleic acid
molecule 13 may be sequenced in parallel or sequentially to confirm
the nucleic acid sequence or to obtain complete sequence data. In
alternative embodiments of the invention, both the nucleic acid
molecule 13 and its complementary strand may be sequenced to
confirm the accuracy of the sequence information.
[0011] In certain embodiments of the invention, the nucleic acid 13
to be sequenced is DNA, although it is contemplated that other
nucleic acids 13 comprising RNA or synthetic nucleotide analogs
could be sequenced as well. The following detailed description
contains numerous specific details in order to provide a more
thorough understanding of the disclosed embodiments of the
invention. However, it will be apparent to those skilled in the art
that the embodiments of the invention may be practiced without
these specific details. In other instances, devices, methods,
procedures, and individual components that are well known in the
art have not been described in detail herein.
[0012] In various embodiments of the invention, exemplified in FIG.
1, nucleotides may be covalently attached to Raman labels to
enhance the Raman signal detected by surface enhanced Raman
spectroscopy (SERS), surface enhanced resonance Raman spectroscopy
(SERRS), coherent anti-Stokes Raman spectroscopy (CARS) or other
known Raman detection techniques. In some embodiments of the
invention, such labeled nucleotides may be incorporated into a
newly synthesized nucleic acid strand 13 using standard nucleic
acid polymerization techniques. Typically, either a primer of
specific sequence or one or more random primers is allowed to
hybridize to a template nucleic acid. Upon addition of a polymerase
and labeled nucleotides, the Raman labeled nucleotides are
covalently attached to the 3' end of the primer, resulting in the
formation of a labeled nucleic acid strand 13 complementary in
sequence to the template.
[0013] After synthesis, the labeled nucleic acid strand 13 may be
digested with one or more exonucleases 15. The skilled artisan will
realize that the disclosed methods are not limited to exonucleases
15 per se, but may utilize any enzyme or other reagent capable of
sequentially removing nucleotides 16, 130 from at least one end of
a nucleic acid 13. In certain embodiments of the invention, Raman
labeled nucleotides 16, 130 are sequentially released from the 3'
end 17 of the labeled nucleic acid 13. After separation from the
labeled nucleic acid 13, the Raman labeled nucleotides 16, 130 are
detected by a detection unit 18, 180, 300. Information on
sequentially detected labeled nucleotides 16, 130 is used to
compile a sequence of the labeled nucleic acid 13, which is
complementary to the sequence of the template strand.
[0014] In some embodiments of the invention, the labeled nucleic
acid strand 13 may be separated from the unlabeled template strand
as well as unincorporated nucleotides prior to exonuclease 15
treatment. This may be accomplished, for example, by using a primer
that has been cross-linked to a surface 14 or that contains biotin
or a similar group that may be attached to a surface 14. Biotin
labeled primers may be attached to a surface 14 that has been
covalently modified with avidin or streptavidin. The labeled
nucleic acid 13 may be separated from the unlabeled template strand
by known techniques.
[0015] In certain embodiments of the invention, each of the four
types of nucleotide may be attached to a distinguishable Raman
label. In other embodiments of the invention, only the purine
nucleotides (cytosine and/or thymine and/or uracil) may be labeled.
In one exemplary embodiment, the labeled nucleotides may comprise
biotin-labeled deoxycytidine-5'-triphosphate (biotin-dCTP) and
digoxigenin-labeled deoxyuridine-5'-triphosphate
(digoxigenin-dUTP).
[0016] In alternative embodiments of the invention, exemplified in
FIG. 2, the Raman signal may be enhanced by covalent attachment of
nucleotides 16, 130 to nanoparticles 140. In certain embodiments of
the invention, such attachment would follow exonuclease 15
treatment of a nucleic acid 13 as disclosed in FIG. 1. In some
embodiments of the invention, the nanoparticles 140 are silver or
gold, but other types of nanoparticles 140 known to provide surface
enhanced Raman signals are contemplated. The nanoparticles 140 may
either be single nanoparticles 140, aggregates of nanoparticles
140, or some mixture of single and aggregated nanoparticles 140. In
certain embodiments of the invention, a linker compound may be used
to attach the nucleotides 16, 130 to the nanoparticles 140. In
various embodiments of the invention, the linker compound may be
between 1 to 100 nanometers (nm), 2 to 90 nm, 3 to 80 nm, 4 to 70
nm, 5 to 60 nm, 10 to 50 nm, 15 to 40 nm or 20 to 30 nm in length.
In certain embodiments of the invention, the linker compound may be
between 1 to 50, 1 to 5, 2 to 10, 10 to 20 nm or about 5 nm in
length. In other embodiments of the invention, two or more
nanoparticles 140 may be attached together using linker
compounds.
[0017] Following covalent attachment, the nanoparticle-nucleotide
complexes 150 may pass through a flow-through cell 170, 290 where
they are detected by SERS, SERRS and/or CARS using a detection unit
18, 180, 300. In some alternative embodiments of the invention, the
nucleotides 16, 130 may be unmodified, while in other alternative
embodiments the nucleotides 16, 130 may be modified with one or
more Raman labels. In certain embodiments of the invention, each
type of nucleotide 16, 130 may be attached to a distinguishable
Raman label. In other embodiments only pyrimidines 16, 130 may be
labeled.
DEFINITIONS
[0018] As used herein, "a" or "an" may mean one or more than one of
an item.
[0019] As used herein, "operably coupled" means that there is a
functional interaction between two or more units. For example, a
detector 21, 310 may be "operably coupled" to a flow-through cell
170, 290 if the detector 21, 310 is arranged so that it may detect
analytes, such as nucleotides 16, 130, as they pass through the
flow-through cell 170, 290.
[0020] "Nucleic acid" 13 encompasses DNA, RNA, single-stranded,
double-stranded or triple stranded and any chemical modifications
thereof. Virtually any modification of the nucleic acid 13 is
contemplated. As used herein, a single stranded nucleic acid 13 may
be denoted by the prefix "ss", a double stranded nucleic acid 13 by
the prefix "ds", and a triple stranded nucleic acid 13 by the
prefix "ts."
[0021] A "nucleic acid" 13 may be of almost any length, from 10,
20, 30, 40, 50, 60, 75, 100, 150, 200, 250, 300, 400, 500, 600,
700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,
5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000,
40,000, 50,000, 75,000, 100,000, 150,000, 200,000, 500,000,
1,000,000, 1,500,000, 2,000,000, 5,000,000 or even more bases in
length, up to a full-length chromosomal DNA molecule 13.
[0022] A "nucleoside" 16, 130 is a molecule comprising a purine or
pyrimidine base (adenine--"A", cytosine--"C", guanine--"G",
thymine--"T" or uracil--"U") or any chemical modification or
structural analog thereof, covalently attached to a pentose sugar
such as deoxyribose, ribose or derivatives or analogs of pentose
sugars.
[0023] A "nucleotide" 16, 130 refers to a nucleoside 16, 130.
further comprising at least one phosphate group covalently attached
to the pentose sugar. In some embodiments of the invention, the
nucleotides 16, 130 are ribonucleoside monophosphates 16, 130 or
deoxyribonucleoside monophosphates 16, 130, although it is
anticipated that nucleoside diphosphates or triphosphates 16, 130
could be produced and detected. In other embodiments of the
invention, nucleosides 16, 130 may be released from the nucleic
acid molecule 13. It is contemplated that various substitutions or
modifications may be made in the structure of the nucleotides 16,
130, so long as they are capable of being incorporated into a
nucleic acid 13 by polymerase activity and released by an
exonuclease 15 or equivalent reagent. In embodiments of the
invention involving one or more labels attached to one or more
types of nucleotide 16, 130, the label may be attached to any
portion of the nucleotide 16, 130, such as the base, the sugar or
the phosphate groups or their analogs, so long as the label does
not interfere with the polymerization and/or digestion of a nucleic
acid 13. The terms "nucleotide" and "labeled nucleotide" encompass,
but are not limited to, all non-naturally nucleotide complexes,
such as nucleotide-nanoparticle complexes and nucleotide-label
complexes.
[0024] A "Raman label" may be any organic or inorganic molecule,
atom, complex or structure capable of producing a detectable Raman
signal, including but not limited to synthetic molecules, dyes,
naturally occurring pigments such as phycoerythrin, organic
nanostructures such as C60, buckyballs and carbon nanotubes, metal
nanostructures such as gold or silver nanoparticles or nanoprisms
and nano-scale semiconductors such as quantum dots. Numerous
examples of Raman labels are disclosed below. The skilled artisan
will realize that such examples are not limiting, and that "Raman
label" encompasses any organic or inorganic atom, molecule,
compound or structure known in the art that can be detected by
Raman spectroscopy.
Nucleic Acids
[0025] Nucleic acid molecules 13 to be sequenced may be prepared by
any technique known in the art. In certain embodiments of the
invention, the nucleic acids 13 are naturally occurring DNA or RNA
molecules. Virtually any naturally occurring nucleic acid 13 may be
prepared and sequenced by the disclosed methods including, without
limit, chromosomal, mitochondrial and chloroplast DNA and
ribosomal, transfer, heterogeneous nuclear and messenger RNA
(mRNA). Methods for preparing and isolating various forms of
nucleic acids 13 are known. (See, e.g., Guide to Molecular Cloning
Techniques, eds. Berger and Kimmel, Academic Press, New York, N.Y.,
1987; Molecular Cloning: A Laboratory Manual, 2nd Ed., eds.
Sambrook, Fritsch and Maniatis, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., 1989). The methods disclosed in the cited
references are exemplary only and any variation known in the art
may be used. In cases where single stranded DNA (ssDNA) 13 is to be
sequenced, an ssDNA 13 may be prepared from double stranded DNA
(dsDNA) by any known method. Such methods may involve heating dsDNA
and allowing the strands to separate, or may alternatively involve
preparation of ssDNA 13 from dsDNA by known amplification or
replication methods, such as cloning into M13. Any such known
method may be used to prepare ssDNA or ssRNA 13.
[0026] Although certain embodiments of the invention concern
preparation of naturally occurring nucleic acids 13, virtually any
type of nucleic acid 13 that can serve as a substrate for an
exonuclease or equivalent reagent 15 could potentially be
sequenced. For example, nucleic acids 13 prepared by various
amplification techniques, such as polymerase chain reaction (PCRTM)
amplification, could be sequenced. (See U.S. Pat. Nos. 4,683,195,
4,683,202 and 4,800,159.) Nucleic acids 13 to be sequenced may
alternatively be cloned in standard vectors, such as plasmids,
cosmids, BACs (bacterial artificial chromosomes) or YACs (yeast
artificial chromosomes). (See, e.g., Berger and Kimmel, 1987;
Sambrook et al., 1989.) Nucleic acid inserts 13 may be isolated
from vector DNA, for example, by excision with appropriate
restriction endonucleases, followed by agarose gel electrophoresis.
Methods for isolation of insert nucleic acids 13 are well
known.
Isolation of Single Nucleic Acid Molecules
[0027] In certain embodiments of the invention, the nucleic acid
molecule 13 to be sequenced is a single molecule of ssDNA or ssRNA.
A variety of methods for selection and manipulation of single
nucleic acid molecules 13 may be used, for example, hydrodynamic
focusing, micro-manipulator coupling, optical trapping, or a
combination of these and similar methods. (See, e.g., Goodwin et
al., 1996, Acc. Chem. Res. 29:607-619; U.S. Pat. Nos. 4,962,037;
5,405,747; 5,776,674; 6,136,543; 6,225,068.)
[0028] In certain embodiments of the invention, microfluidics or
nanofluidics may be used to sort and isolate nucleic acid molecules
13. Hydrodynamics may be used to manipulate the movement of nucleic
acids 13 into a microchannel, microcapillary, or a micropore. In
one embodiment of the invention, hydrodynamic forces may be used to
move nucleic acid molecules 13 across a comb structure to separate
single nucleic acid molecules 13. Once the nucleic acid molecules
13 have been separated, hydrodynamic focusing may be used to
position the molecules 13 within a reaction chamber 11, 220. A
thermal or electric potential, pressure or vacuum can also be used
to provide a motive force for manipulation of nucleic acids 13. In
exemplary embodiments of the invention, manipulation of nucleic
acids 13 for sequencing may involve the use of a channel block
design incorporating microfabricated channels and an integrated gel
material (see U.S. Pat. Nos. 5,867,266 and 6,214,246).
[0029] In another embodiment of the invention, a sample containing
the nucleic acid molecule 13 may be diluted prior to coupling to an
immobilization surface 14. In exemplary embodiments of the
invention, the immobilization surface 14 may be in the form of
magnetic or non-magnetic beads or other discrete structural units.
At an appropriate dilution, each bead 14 will have a statistical
probability of binding zero or one nucleic acid molecule 13. Beads
14 with one attached nucleic acid molecule 13 may be identified
using, for example, fluorescent dyes and flow cytometer sorting or
magnetic sorting. Depending on the relative sizes and uniformity of
the beads 14 and the nucleic acids 13, it may be possible to use a
magnetic filter and mass separation to separate beads 14 containing
a single bound nucleic acid molecule 13. In other embodiments of
the invention, multiple nucleic acids 13 attached to a single bead
or other immobilization surface 14 may be sequenced.
[0030] In alternative embodiments of the invention, a coated fiber
tip 14 may be used to generate single molecule nucleic acids 13 for
sequencing (e.g., U.S. Pat. No. 6,225,068). In other alternative
embodiments, the immobilization surfaces 14 may be prepared to
contain a single molecule of avidin or other cross-linking agent.
Such a surface 14 could attach a single biotinylated nucleic acid
molecule 13 to be sequenced. This embodiment is not limited to the
avidin-biotin binding system, but may be adapted to any known
coupling system.
[0031] In other alternative embodiments of the invention, an
optical trap may be used for manipulation of single molecule
nucleic acid molecules 13 for sequencing. (e.g., U.S. Pat. No.
5,776,674). Exemplary optical trapping systems are commercially
available from Cell Robotics, Inc. (Albuquerque, N.Mex.), S+L GmbH
(Heidelberg, Germany) and P.A.L.M. GmbH (Wolfratshausen,
Germany).
Raman Labels
[0032] Certain embodiments of the invention may involve attaching a
label to the nucleotides 16, 130 to facilitate their measurement by
the detection unit 18, 180, 300. Non-limiting examples of labels
that could be used for Raman spectroscopy include TRIT (tetramethyl
rhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red
dye, phthalic acid, terephthalic acid, isophthalic acid, cresyl
fast violet, cresyl blue violet, brilliant cresyl blue,
para-aminobenzoic acid, erythrosine, biotin, digoxigenin,
5-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein,
5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein,
5-carboxy rhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino
phthalocyanines, azomethines, cyanines, xanthines,
succinylfluoresceins and aminoacridine. These and other Raman
labels may be obtained from commercial sources (e.g., Molecular
Probes, Eugene, Oreg.).
[0033] Polycyclic aromatic compounds may function as Raman labels,
as is known in the art. Other labels that may be of use for
particular embodiments of the invention include cyanide, thiol,
chlorine, bromine, methyl, phosphorus and sulfur. In certain
embodiments of the invention, carbon nanotubes may be of use as
Raman labels. The use of labels in Raman spectroscopy is known
(e.g., U.S. Pat. Nos. 5,306,403 and 6,174,677). The skilled artisan
will realize that the Raman labels used should generate
distinguishable Raman spectra and may be specifically bound to or
associated with different types of nucleotides 16, 130.
[0034] Labels may be attached directly to the nucleotides 16, 130
or may be attached via various linker compounds. Cross-linking
reagents and linker compounds of use in the disclosed methods are
further described below. Alternatively, nucleotides that are
covalently attached to Raman labels are available from standard
commercial sources (e.g., Roche Molecular Biochemicals,
Indianapolis, Ind.; Promega Corp., Madison, Wis.; Ambion, Inc.,
Austin, Tex.; Amersham Pharmacia Biotech, Piscataway, N.J.). Raman
labels that contain reactive groups designed to covalently react
with other molecules, such as nucleotides 16, 130, are commercially
available (e.g., Molecular Probes, Eugene, Oreg.). Methods for
preparing labeled nucleotides and incorporating them into nucleic
acids 13 are known (e.g., U.S. Pat. Nos. 4,962,037; 5,405,747;
6,136,543; 6,210,896).
Nanoparticles
[0035] Certain embodiments of the invention involve the use of
nanoparticles 140 to enhance the Raman signal obtained from
nucleotides 16, 130. In some embodiments of the invention, the
nanoparticles 140 are silver or gold nanoparticles 140, although
any nanoparticles 140 capable of providing a surface enhanced Raman
spectroscopy (SERS) signal may be used. In alternative embodiments
of the invention, the nanoparticles 140 may be nanoprisms (Jin et
al., Science 294:1902-3, 2001.) In various embodiments of the
invention, nanoparticles 140 of between 1 nm and 2 micrometers
(.mu.m) in diameter may be used. In alternative embodiments of the
invention, nanoparticles 140 of between 2 nm to 1 .mu.m, 5 nm to
500 nm, 10 nm to 200 nm, 20 nm to 100 nm, 30 nm to 80 nm, 40 nm to
70 nm or 50 to 60 nm diameter are contemplated. In certain
embodiments of the invention, nanoparticles 140 with an average
diameter of 10 to 50 nm, 50 to 100 nm or about 100 nm are
contemplated. The nanoparticles 140 may be approximately spherical,
rod-like, edgy, faceted or pointy in shape, although nanoparticles
140 of any shape or of irregular shape may be used. Methods of
preparing nanoparticles are known (e.g., U.S. Pat. Nos. 6,054,495;
6,127,120; 6,149,868; Lee and Meisel, J. Phys. Chem. 86:3391-3395,
1982; Jin et al., 2001). Nanoparticles may also be obtained from
commercial sources (e.g., Nanoprobes Inc., Yaphank, N.Y.;
Polysciences, Inc., Warrington, Pa.).
[0036] In certain embodiments of the invention, the nanoparticles
140 may be single nanoparticles 140 and/or random aggregates of
nanoparticles 140 (colloidal nanoparticles 140). In other
embodiments of the invention, nanoparticles 140 may be cross-linked
to produce particular aggregates of nanoparticles 140, such as
dimers, trimers, tetramers or other aggregates. Certain alternative
embodiments of the invention may use heterogeneous mixtures of
aggregates of different size, while other alternative embodiments
may use homogenous populations of nanoparticles 140. In certain
embodiments of the invention, aggregates containing a selected
number of nanoparticles 140 (dimers, trimers, etc.) may be enriched
or purified by known techniques, such as ultracentrifugation in
sucrose solutions. In various embodiments of the invention,
nanoparticle 140 aggregates of about 100, 200, 300, 400, 500, 600,
700, 800, 900 to 1000 nm in size or larger are contemplated.
[0037] Methods of cross-linking nanoparticles 140 are known (e.g.,
Feldheim, "Assembly of metal nanoparticle arrays using molecular
bridges," The Electrochemical Society Interface, Fall, 2001, pp.
22-25). Gold nanoparticles 140 may be cross-linked, for example,
using bifunctional linker compounds bearing terminal thiol or
sulfhydryl groups. Upon reaction with gold nanoparticles 140, the
linker forms nanoparticle 140 dimers that are separated by the
length of the linker. In other embodiments of the invention,
linkers with three, four or more thiol groups may be used to
simultaneously attach to multiple nanoparticles 140 (Feldheim,
2001). The use of an excess of nanoparticles 140 to linker
compounds prevents formation of multiple cross-links and
nanoparticle 140 precipitation. Aggregates of silver nanoparticles
140 may be formed by standard synthesis methods known in the
art.
[0038] In alternative embodiments of the invention, the
nanoparticles 140 may be modified to contain various reactive
groups before they are attached to linker compounds. Modified
nanoparticles 140 are commercially available, such as Nanogold.RTM.
nanoparticles 140 from Nanoprobes, Inc. (Yaphank, N.Y.).
Nanogold.RTM. nanoparticles 140 may be obtained with either single
or multiple maleimide, amine or other groups attached per
nanoparticle 140. The Nanogold.RTM. nanoparticles 140 are also
available in either positively or negatively charged form. Such
modified nanoparticles 140 may be attached to a variety of known
linker compounds to provide dimers, trimers or other aggregates of
nanoparticles 140.
[0039] The type of linker compound used is not limiting, so long as
it results in the production of small aggregates of nanoparticles
140 that will not precipitate in solution. In some embodiments of
the invention, the linker group may comprise phenylacetylene
polymers (Feldheim, 2001). Alternatively, linker groups may
comprise polytetrafluoroethylene, polyvinyl pyrrolidone,
polystyrene, polypropylene, polyacrylamide, polyethylene or other
known polymers. The linker compounds of use are not limited to
polymers, but may also include other types of molecules such as
silanes, alkanes, derivatized silanes or derivatized alkanes.
[0040] In various embodiments of the invention, the nanoparticles
140 may be covalently attached to nucleotides 16, 130. In
alternative embodiments of the invention, the nucleotides 16, 130
may be directly attached to the nanoparticles 140, or may be
attached to linker compounds that are covalently or non-covalently
bonded to the nanoparticles 140. In such embodiments of the
invention, rather than cross-linking two or more nanoparticles 140
together the linker compounds may be used to attach a nucleotide
16, 130 to a nanoparticle 140 or a nanoparticle 140 aggregate. In
particular embodiments of the invention, the nanoparticles 140 may
be coated with derivatized silanes. Such modified silanes may be
covalently attached to nucleotides 16, 130 using standard methods.
Various methods known for cross-linking nucleic acids 13 to
surfaces 14 discussed below may also be used to attach nucleotides
16, 130 to nanoparticles 140. It is contemplated that the linker
compounds used to attach nucleotides 16, 130 may be of almost any
length, ranging from about 0.05, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 27, 30, 35, 40, 45, 50, 55, 60, 65, 60, 80, 90 to 100
nm or even greater length. Certain embodiments of the invention may
use linkers of heterogeneous length.
[0041] In other embodiments of the invention, nucleotides 16, 130
may be adsorbed on the surface of the nanoparticles 140 or may be
in close proximity to the nanoparticles 140 (between about 0.2 and
1.0 nm). The skilled artisan will realize that it covalent
attachment of the nucleotides 16, 130 to nanoparticles 140 is not
required in order to generate an enhanced Raman signal by SERS,
SERRS or CARS.
[0042] In the exemplary embodiment of the invention disclosed in
FIG. 2, the nucleotides 130 are attached to nanoparticles 140 as
they travel down a microfluidic channel 160 to form
nucleotide-nanoparticle complexes 150. In certain embodiments of
the invention, the length of time available for the cross-linking
reaction to occur may be very limited. Such embodiments may utilize
highly reactive cross-linking groups with rapid reaction rates,
such as epoxide groups, azido groups, arylazido groups, triazine
groups or diazo groups. In certain embodiments of the invention,
the cross-linking groups may be photoactivated by exposure to
intense light, such as a laser. For example, photoactivation of
diazo or azido compounds results in the formation, respectively, of
highly reactive carbene and nitrene moieties. In certain
embodiments of the invention, the reactive groups may be selected
so that they can only attach the nanoparticles 140 to nucleotides
16, 130, rather than cross-linking the nanoparticles 140 to each
other. The selection and preparation of reactive cross-linking
groups capable of binding to nucleotides 16, 130 is known in the
art. In alternative embodiments of the invention, nucleotides 16,
130 may themselves be covalently modified, for example with a
sulfhydryl group that can attach to gold nanoparticles 140.
[0043] In certain embodiments of the invention, nanoparticles 140
may be manipulated into microfluidic channels 120, 160, 270, 280 by
any method known in the art, such as microfluidics, nanofluidics,
hydrodynamic focusing or electro-osmosis. In some embodiments of
the invention, use of charged linker compounds or charged
nanoparticles 140 may facilitate manipulation of nanoparticles 140
through the use of electrical gradients.
Immobilization of Nucleic Acids
[0044] In certain embodiments of the invention, as exemplified in
FIG. 1, one or more nucleic acid molecules 13 may be attached to a
surface 14 such as functionalized glass, silicon, silicate, PDMS
(polydimethyl siloxane), polyvinylidene difluoride (PVDF), silver
or other metal coated surfaces, quartz, plastic, PTFE
(polytetrafluoroethylene), PVP (polyvinyl pyrrolidone), poly(vinyl
chloride), poly(methyl methacrylate), poly(dimethyl siloxane),
polystyrene, polypropylene, polyacrylamide, latex, nylon,
nitrocellulose, glass beads, magnetic beads, photopolymers which
contain photoreactive species such as nitrenes, carbenes and ketyl
radicals capable of forming covalent links with nucleic acid
molecules 13 (See U.S. Pat. Nos. 5,405,766 and 5,986,076) or any
other material known in the art that is capable of having
functional groups such as amino, carboxyl, thiol, hydroxyl or
Diels-Alder reactants incorporated on its surface 14.
[0045] In some embodiments of the invention, the surface functional
groups may be covalently attached to cross-linking compounds so
that binding interactions between nucleic acid molecule 13 and
exonuclease 15 and/or polymerase may occur without steric
hindrance. Typical cross-linking groups include ethylene glycol
oligomers and diamines. Attachment may be by either covalent or
non-covalent binding. Various methods of attaching nucleic acid
molecules 13 to surfaces 14 are known in the art and may be
employed. In certain embodiments of the invention, the nucleic acid
molecule 13 is fixed in place and immersed in a microfluidic flow
down a flow path 12 and/or microfluidic channel 110, 160, 260, 280
that transports the released nucleotides 16, 130 past a detection
unit 18, 180, 300. In non-limiting examples, the microfluidic flow
may result from a bulk flow of solvent down a flow path 12 and/or
microfluidic channel 110, 160, 260, 280.
[0046] In alternative embodiments of the invention, the bulk medium
moves only slowly or not at all, but charged species within the
solution (such as negatively charged nucleotides 16, 130) move down
a flow path 12 and/or microfluidic channel 110, 160, 260, 280 in
response to an externally applied electrical field.
[0047] Immobilization of nucleic acid molecules 13 may be achieved
by a variety of known methods. In an exemplary embodiment of the
invention, immobilization may be achieved by coating a surface 14
with streptavidin or avidin and the subsequent attachment of a
biotinylated nucleic acid 13 (Holmstrom et al., Anal. Biochem.
209:278-283, 1993). Immobilization may also occur by coating a
silicon, glass or other surface 14 with poly-L-Lys (lysine) or poly
L-Lys, Phe (phenylalanine), followed by covalent attachment of
either amino- or sulfhydryl-modified nucleic acids 13 using
bifunctional crosslinking reagents (Running et al., BioTechniques
8:276-277, 1990; Newton et al., Nucleic Acids Res. 21:1155-62,
1993). Amine residues may be coated on a surface 14 through the use
of aminosilane.
[0048] Immobilization may take place by direct covalent attachment
of 5'-phosphorylated nucleic acids 13 to chemically modified
surfaces 14 (Rasmussen et al., Anal. Biochem. 198:138-142, 1991).
The covalent bond between the nucleic acid 13 and the surface 14
may be formed by condensation with a water-soluble carbodiimide.
This method facilitates a predominantly 5'-attachment of the
nucleic acids 13 via their 5'-phosphates.
[0049] DNA 13 is commonly bound to glass by first silanizing the
glass surface 14, then activating with carbodiimide or
glutaraldehyde. Alternative procedures may use reagents such as
3-glycidoxypropyltrimethoxysilane (GOP) or
aminopropyltrimethoxysilane (APTS) with DNA 13 linked via amino
linkers incorporated at either the 3' or 5' end of the molecule.
DNA 13 may be bound directly to membrane surfaces 14 using
ultraviolet radiation. Other non-limiting examples of
immobilization techniques for nucleic acids 13 are disclosed in
U.S. Pat. Nos. 5,610,287, 5,776,674 and 6,225,068.
[0050] Bifunctional cross-linking reagents may be of use in various
embodiments of the invention, such as attaching a nucleic acid
molecule 13 to a surface 14. The bifunctional cross-linking
reagents can be divided according to the specificity of their
functional groups, e.g., amino, guanidino, indole, or carboxyl
specific groups. Exemplary methods for cross-linking molecules are
disclosed in U.S. Pat. Nos. 5,603,872 and 5,401,311. Cross-linking
reagents include glutaraldehyde (GAD), biftnctional oxirane (OXR),
ethylene glycol diglycidyl ether (EGDE), and carbodiumides, such as
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
Nucleic Acid Synthesis
Polymerases
[0051] Certain embodiments of the invention involve binding of a
synthetic reagent, such as a DNA polymerase, to a primer molecule
and the addition of Raman labeled nucleotides to the 3' end of the
primer. Non-limiting examples of polymerases include DNA
polymerases, RNA polymerases, reverse transcriptases, and
RNA-dependent RNA polymerases. The differences between these
polymerases in terms of their "proofreading" activity and
requirement or lack of requirement for primers and promoter
sequences are known in the art. Where RNA polymerases are used as
the polymerase, a template molecule to be sequenced may be
double-stranded DNA. Non-limiting examples of polymerases include
Thermotoga maritima DNA polymerase, AmplitaqFS3 DNA polymerase,
Taquenase3 DNA polymerase, ThermoSequenase3, Taq DNA polymerase,
Qbeta3 replicase, T4 DNA polymerase, Thermus thermophilus DNA
polymerase, RNA-dependent RNA polymerase and SP6 RNA
polymerase.
[0052] A number of polymerases are commercially available,
including Pwo DNA Polymerase (Boehringer Mannheim Biochemicals,
Indianapolis, Ind.); Bst Polymerase (Bio-Rad Laboratories,
Hercules, Calif.); IsoTherm3 DNA Polymerase (Epicentre
Technologies, Madison, Wis.); Moloney Murine Leukemia Virus Reverse
Transcriptase, Pfu DNA Polymerase, Avian Myeloblastosis Virus
Reverse Transcriptase, Thermus flavus (Tfl) DNA Polymerase and
Thermococcus litoralis (Tli) DNA Polymerase (Promega Corp.,
Madison, Wis); RAV2 Reverse Transcriptase, HIV-1 Reverse
Transcriptase, T7 RNA Polymerase, T3 RNA Polymerase, SP6 RNA
Polymerase, E. coli RNA Polymerase, Thermus aquaticus DNA
Polymerase, T7 DNA Polymerase .+-.3'.fwdarw.5' exonuclease, Klenow
Fragment of DNA Polymerase I, Thermus `ubiquitous` DNA Polymerase,
and DNA polymerase I (Amersham Pharmacia Biotech, Piscataway,
N.J.). Any polymerase known in the art capable of template
dependent polymerization of labeled nucleotides may be used. (See,
e.g., Goodman and Tippin, Nat. Rev. Mol. Cell Biol. 1(2):101-9,
2000; U.S. Pat. No. 6,090,589.) Methods of using polymerases to
synthesize nucleic acids 13 from labeled nucleotides are known
(e.g., U.S. Pat. Nos. 4,962,037; 5,405,747; 6,136,543;
6,210,896).
Primers
[0053] Generally, primers are between ten and twenty bases in
length, although longer primers may be employed. In certain
embodiments of the invention, primers are designed to be
complementary in sequence to a known portion of a template nucleic
acid molecule. Known primer sequences may be used, for example,
where primers are selected for identifying sequence variants
adjacent to known constant chromosomal sequences, where an unknown
nucleic acid sequence is inserted into a vector of known sequence,
or where a native nucleic acid has been partially sequenced. Other
embodiments of the invention involve sequencing a nucleic acid 13
in the absence of a known primer-binding site. In such cases, it
may be possible to use random primers, such as random hexamers or
random oligomers to initiate polymerization. Methods for synthesis
of primers of any sequence are known.
Nucleic Acid Digestion
[0054] In certain embodiments of the invention, exemplified in FIG.
1, methods of nucleic acid 13 sequencing involve binding of an
exonuclease 15 or equivalent reagent to the free end 17 of a
nucleic acid molecule 13 and removal of nucleotides 16, 130 one at
a time. Non-limiting examples of nucleic acid digesting enzymes 15
of potential use include E. coli exonuclease I, III, V or VII, Bal
31 exonuclease, mung bean nuclease, S1 nuclease, E. coli DNA
polymerase I holoenzyme or Klenow fragment, RecJ, exonuclease T, T4
or T7 DNA polymerase, Taq polymerase, exonuclease T7 gene 6, snake
venom phosphodiesterase, spleen phosphodiesterase, Thermococcus
litoralis DNA polymerase, Pyrococcus sp. GB-D DNA polymerase,
lambda exonuclease, S. aureus micrococcal nuclease, DNase I,
ribonuclease A, T1 micrococcal nuclease, or other exonucleases
known in the art. Exonucleases 15 are available from commercial
sources such as New England Biolabs (Beverly, Mass.), Amersham
Pharmacia Biotech (Piscataway, N.J.), Promega (Madison, Wis.),
Sigma Chemicals (St. Louis, Mo.) or Boehringer Mannheim
(Indianapolis, Ind.).
[0055] The skilled artisan will realize that enzymes with
exonuclease 15 activity may remove nucleotides 16, 130 from the 5'
end, the 3' end, or either end of nucleic acid molecules 13. They
can show specificity for RNA, DNA or both RNA and DNA 13. Their
activity may depend on the use of either single or double-stranded
nucleic acids 13. They may be differentially affected by salt
concentration, temperature, pH, or divalent cations. These and
other properties of exonucleases 15 are known in the art. In
certain embodiments of the invention, the rate of exonuclease 15
activity may be manipulated to coincide with the optimal rate of
analysis of nucleotides 16, 130 by the detection unit 18, 180, 300.
Various methods are known for adjusting the rate of exonuclease 15
activity, including adjusting the temperature, pressure, pH, salt
or divalent cation concentration in a reaction chamber 11, 220.
[0056] Although nucleoside monophosphates 16, 130 will generally be
released from nucleic acids 13 by exonuclease 15 activity, the
embodiments of the invention are not limited to detection of any
particular form of free nucleotide or nucleoside 16, 130 but
encompass any monomer 16, 130 that may be released from a nucleic
acid 13.
Reaction Chamber and Integrated Chip
[0057] As exemplified in FIG. 1, some embodiments of the invention
concern apparatus 10, 100, 210 comprising a reaction chamber 11,
220 designed to contain an immobilization surface 14, nucleic acid
molecule 13, exonuclease 15 and nucleotides 16, 130 in an aqueous
environment. In some embodiments of the invention, the reaction
chamber 11, 220 may be temperature controlled, for example by
incorporation of Pelletier elements or other methods known in the
art. Methods of controlling temperature for low volume liquids are
known. (See, e.g., U.S. Pat. Nos. 5,038,853, 5,919,622, 6,054,263
and 6,180,372.)
[0058] In certain embodiments of the invention, the reaction
chamber 11, 220 and any associated fluid channels, for example, a
flow path 12, microfluidic channels 110, 160, 260, 280 or channels
120, 230, 240, 270, 350, 360 to provide connections to waste ports,
to a nucleic acid 13 loading port, to a nanoparticle reservoir 370,
to a source of exonuclease 15 or other fluid compartments are
manufactured in a batch fabrication process, as known in the fields
of computer chip manufacture and/or microcapillary chip
manufacture. In some embodiments of the invention, the reaction
chamber 11, 220 and other components of the apparatus 10, 100, 210,
such as the flow path 12 and/or microfluidic channels 120, 160,
260, 280 may be manufactured as a single integrated chip. Such a
chip may be manufactured by methods known in the art, such as by
photolithography and etching. However, the manufacturing method is
not limiting and other methods known in the art may be used, such
as laser ablation, injection molding, casting, molecular beam
epitaxy, dip-pen nanolithograpy, chemical vapor deposition (CVD)
fabrication, electron beam or focused ion beam technology or
imprinting techniques. Methods for manufacture of
nanoelectromechanical systems may be used for certain embodiments
of the invention. (See, e.g., Craighead, Science 290:1532-36,
2000.) Microfabricated chips are commercially available from, e.g.,
Caliper Technologies Inc. (Mountain View, Calif.) and ACLARA
BioSciences Inc. (Mountain View, Calif.).
[0059] To facilitate detection of nucleotides 16, 130 by the
detection unit 18, 180, 300 the material comprising the flow path
12 or flow-through cell 170, 290 may be selected to be transparent
to electromagnetic radiation at the excitation and emission
frequencies used for the detection unit 18, 180, 300. Glass,
silicon, and any other materials that are generally transparent in
the wavelengths used for Raman spectroscopy may be used. In some
embodiments of the invention the surfaces of the flow path 12 or
flow-through cell 170, 290 that are opposite the detection unit 18,
180, 300 may be coated with silver, gold, platinum, copper,
aluminum or other materials that are relatively opaque to the
detection unit 18, 180, 300. In that position, the opaque material
is available to enhance the Raman signal, for example by SERS,
while not interfering with the function of the detection unit 18,
180, 300. Alternatively, the flow path 12 or flow-through cell 170,
290 may contain a mesh comprising silver, gold, platinum, copper,
aluminum or other Raman signal enhancing metal.
Flow Path and Microfluidic Channels
[0060] In certain embodiments of the invention, the nucleotides 16,
130 released from a nucleic acid 13 are moved down a flow path 12
and/or microfluidic channels 110, 160, 260, 280 past a detection
unit 18, 180, 300. A non-limiting example of techniques for
transport of nucleotides 16, 130 includes microfluidic techniques.
The flow path 12 and/or microfluidic channels 110, 160, 260, 280
can comprise a microcapillary (e.g., from ACLARA BioSciences Inc.,
Mountain View, Calif.) or a liquid integrated circuit (e.g.,
Caliper Technologies Inc., Mountain View, Calif.).
[0061] In certain embodiments of the invention, the nucleotides 16,
130 to be detected move down the flow path 12 and/or microfluidic
channels 110, 160, 260, 280 by bulk flow of solvent. In other
embodiments of the invention, microcapillary electrophoresis may be
used to transport nucleotides 16, 130 down the flow path 12 and/or
microfluidic channels 110, 160, 260, 280. Microcapillary
electrophoresis generally involves the use of a thin capillary or
channel that may or may not be filled with a particular separation
medium. Electrophoresis of appropriately charged molecular species,
such as negatively charged nucleotides 16, 130, occurs in response
to an imposed electrical field, negative on the reaction chamber
11, 220 side of the apparatus 10, 100, 210 and positive on the
detection unit 18, 180, 300 side. Although electrophoresis is often
used for size separation of a mixture of components that are
simultaneously added to the microcapillary, it can also be used to
transport similarly sized nucleotides 16, 130 that are sequentially
released from a nucleic acid 13. Because the purine nucleotides (A,
G) 16, 130 are larger than the pyrimidine nucleotides (C, T, U) 16,
130 and would therefore migrate more slowly, the length of the flow
path 12 and/or microfluidic channels 110, 160, 260, 280 and the
corresponding transit time past the detection unit 18, 180, 300 may
kept to a minimum to prevent differential migration from mixing up
the order of nucleotides 16, 130 released from the nucleic acid 13.
Alternatively, the medium filling the microcapillary may be
selected so that the migration rates of purine and pyrimidine
nucleotides 16, 130 down the flow path 12 and/or microfluidic
channels 110, 160, 260, 280 are similar or identical. Methods of
microcapillary electrophoresis have been disclosed, for example, by
Woolley and Mathies (Proc. Natl. Acad. Sci. USA 91:11348-352,
1994).
[0062] In certain embodiments of the invention, flow paths 12
and/or microfluidic channels 110, 160, 260, 280 may contain aqueous
solutions with relatively high viscosity, such as glycerol
solutions. Such high viscosity solutions may serve to decrease the
flow rate and increase the reaction time available, for example,
for cross-linking nucleotides 16, 130 to nanoparticles 140.
[0063] Microfabrication of microfluidic devices, including
microcapillary electrophoretic devices has been disclosed in, e.g.,
Jacobsen et al. (Anal. Biochem, 209:278-283, 1994); Effenhauser et
al. (Anal. Chem. 66:2949-2953, 1994); Harrison et al. (Science
261:895-897, 1993) and U.S. Pat. No. 5,904,824. These methods may
comprise micromolding techniques with silicon masters made using
standard photolithography or focused ion beam techniques, or
photolithographic etching of micron scale channels on silica,
silicon or other crystalline substrates or chips. Such techniques
may be readily adapted for use in the disclosed methods and
apparatus. In some embodiments of the invention, the microcapillary
may be fabricated from the same materials used for fabrication of a
reaction chamber 11, 220, using techniques known in the art.
Detection Unit
[0064] In various embodiments of the invention, the detection unit
18, 180, 300 is designed to detect and quantify nucleotides 16, 130
by Raman spectroscopy. Methods for detection of nucleotides 16, 130
by Raman spectroscopy are known in the art. (See, e.g., U.S. Pat.
Nos. 5,306,403; 6,002,471; 6,174,677). Variations on surface
enhanced Raman spectroscopy (SERS), surface enhanced resonance
Raman spectroscopy (SERRS) and coherent anti-Stokes Raman
spectroscopy (CARS) have been disclosed. The sensitivity of Raman
detection is enhanced by a factor of 106 or more for molecules
adjacent to roughened metal surfaces, such as silver, gold,
platinum, copper or aluminum surfaces.
[0065] A non-limiting example of a Raman detection unit 18, 180,
300 is disclosed in U.S. Pat. No. 6,002,471. An excitation beam 20,
330 is generated by either a frequency doubled Nd:YAG laser 19, 320
at 532 nm wavelength or a frequency doubled Ti:sapphire laser 19,
320 at 365 nm wavelength. Pulsed laser beams 20, 330 or continuous
laser beams 20, 330 may be used. The excitation beam 20, 330 passes
through confocal optics and a microscope objective, and is focused
onto the flow path 12 and/or the flow-through cell 170, 290. The
Raman emission light from the nucleotides 16, 130 is collected by
the microscope objective and the confocal optics and is coupled to
a monochromator for spectral dissociation. The confocal optics
includes a combination of dichroic filters, barrier filters,
confocal pinholes, lenses, and mirrors for reducing the background
signal. Standard full field optics can be used as well as confocal
optics. The Raman emission signal is detected by a Raman detector
21, 310, comprising an avalanche photodiode interfaced with a
computer for counting and digitization of the signal.
[0066] Another example of a Raman detection unit 18, 180, 300 is
disclosed in U.S. Pat. No. 5,306,403, including a Spex Model 1403
double-grating spectrophotometer 21, 310 with a gallium-arsenide
photomultiplier tube (RCA Model C3 1034 or Burle Industries Model
C3103402) operated in the single-photon counting mode. The
excitation source 19, 320 comprises a 514.5 nm line argon-ion laser
19, 320 from SpectraPhysics, Model 166, and a 647.1 nm line of a
krypton-ion laser 19, 320 (Innova 70, Coherent).
[0067] Alternative excitation sources 19, 320 include a nitrogen
laser 19, 320 (Laser Science Inc.) at 337 nm and a helium-cadmium
laser 19, 320 (Liconox) at 325 nm (U.S. Pat. No. 6,174,677), a
light emitting diode 19, 320, an Nd:YLF laser 19, 320, and/or
various ions lasers 19, 320 and/or dye lasers 19, 320. The
excitation beam 20, 330 may be spectrally purified with a bandpass
filter (Corion) and may be focused on the flow path 12 and/or
flow-through cell 170, 290 using a 6.times. objective lens
(Newport, Model L6X). The objective lens may be used to both excite
the nucleotides 16, 130 and to collect the Raman signal, by using a
holographic beam splitter (Kaiser Optical Systems, Inc., Model HB
647-26N18) to produce a right-angle geometry for the excitation
beam 20, 330 and the emitted Raman signal. A holographic notch
filter (Kaiser Optical Systems, Inc.) may be used to reduce
Rayleigh scattered radiation. Alternative Raman detectors 21, 310
include an ISA HR-320 spectrograph equipped with a red-enhanced
intensified charge-coupled device (RE-ICCD) detection system
(Princeton Instruments). Other types of detectors 21, 310 may be
used, such as Fourier-transform spectrographs (based on Michaelson
interferometers), charged injection devices, photodiode arrays,
InGaAs detectors, electron-multiplied CCD, intensified CCD and/or
phototransistor arrays.
[0068] Any suitable form or configuration of Raman spectroscopy or
related techniques known in the art may be used for detection of
nucleotides 16, 130, including but not limited to normal Raman
scattering, resonance Raman scattering, surface enhanced Raman
scattering, surface enhanced resonance Raman scattering, coherent
anti-Stokes Raman spectroscopy (CARS), stimulated Raman scattering,
inverse Raman spectroscopy, stimulated gain Raman spectroscopy,
hyper-Raman scattering, molecular optical laser examiner (MOLE) or
Raman microprobe or Raman microscopy or confocal Raman
microspectrometry, three-dimensional or scanning Raman, Raman
saturation spectroscopy, time resolved resonance Raman, Raman
decoupling spectroscopy or UV-Raman microscopy.
Information Processing and Control System and Data Analysis
[0069] In certain embodiments of the invention, the nucleic acid
sequencing apparatus 10, 100, 210 may comprise an information
processing system. The disclosed methods and apparatus 10, 100, 210
are not limiting for the type of information processing system
used. An exemplary information processing system may incorporate a
computer comprising a bus for communicating information and a
processor for processing information. In one embodiment of the
invention, the processor is selected from the Pentium.RTM. family
of processors, including without limitation the Pentium.RTM. II
family, the Pentium.RTM. III family and the Pentium.RTM. 4 family
of processors available from Intel Corp. (Santa Clara, Calif.). In
alternative embodiments of the invention, the processor may be a
Celeron.RTM., an Itanium.RTM., or a Pentium Xeon.RTM. processor
(Intel Corp., Santa Clara, Calif.). In various other embodiments of
the invention, the processor may be based on Intel.RTM.
architecture, such as Intel.RTM. IA-32 or Intel.RTM. IA-64
architecture. Alternatively, other processors may be used. The
information processing and control system may further comprise any
peripheral devices known in the art, such as memory, display,
keyboard and/or other devices.
[0070] In particular embodiments of the invention, the detection
unit 18, 180, 300 may be operably coupled to the information
processing system. Data from the detection unit 18, 180, 300 may be
processed by the processor and data stored in memory. Data on
emission profiles for standard nucleotides 16, 130 may also be
stored in memory. The processor may compare the emission spectra
from nucleotides 16, 130 in the flow path 12 and/or flow-through
cell 170, 290 to identify the type of nucleotide 16, 130 released
from the nucleic acid molecule 13. The memory may also store the
sequence of nucleotides 16, 130 released from the nucleic acid
molecule 13. The processor may analyze the data from the detection
unit 18, 180, 300 to determine the sequence of the nucleic acid 13.
The information processing system may also perform standard
procedures such as subtraction of background signals and
"base-calling" determination when overlapping signals are
detected.
[0071] While the disclosed methods may be performed under the
control of a programmed processor, in alternative embodiments of
the invention, the methods may be fully or partially implemented by
any programmable or hardcoded logic, such as Field Programmable
Gate Arrays (FPGAs), TTL logic, or Application Specific Integrated
Circuits (ASICs). Additionally, the disclosed methods may be
performed by any combination of programmed general purpose computer
components and/or custom hardware components.
[0072] Following the data gathering operation, the data will
typically be reported to a data analysis operation. To facilitate
the analysis operation, the data obtained by the detection unit 18,
180, 300 will typically be analyzed using a digital computer such
as that described above. Typically, the computer will be
appropriately programmed for receipt and storage of the data from
the detection unit 18, 180, 300 as well as for analysis and
reporting of the data gathered.
[0073] In certain embodiments of the invention, custom designed
software packages may be used to analyze the data obtained from the
detection unit 18, 180, 300. In alternative embodiments of the
invention, data analysis may be performed, using an information
processing system and publicly available software packages.
Non-limiting examples of available software for DNA sequence
analysis include the PRISM3 DNA Sequencing Analysis Software
(Applied Biosystems, Foster City, Calif.), the Sequencher3 package
(Gene Codes, Ann Arbor, Mich.), and a variety of software packages
available through the National Biotechnology Information Facility
at website www.nbif.org/links/1.4.1.phy.
EXAMPLES
Example 1
Nucleic Acid Sequencing Using Raman Labeled Nucleotides
[0074] Certain embodiments of the invention, exemplified in FIG. 1,
involve sequencing of individual single-stranded nucleic acid
molecules 13 that are attached to an immobilization surface 14 in a
reaction chamber 11, 220 and disassembled in a deconstruction
reaction. In such embodiments of the invention, the reaction
chamber 11, 220 contains one or more exonucleases 15 that
sequentially remove one nucleotide 16, 130 at a time from the
unattached end 17 of the nucleic acid molecule 13.
[0075] As the nucleotides 16, 130 are released, they move down a
flow path 12 past a detection unit 18, 180, 300. The detection unit
18, 180, 300 comprises an excitation source 19, 320, such as a
laser, that emits an excitatory beam 20, 330. The excitatory beam
20, 330 interacts with the released nucleotides 16, 130 so that
electrons are excited to a higher energy state. The Raman emission
spectrum that results from the return of the electrons to a lower
energy state is detected by a Raman spectroscopic detector 21, 310,
such as a spectrometer, a monochromator or a charge coupled device
(CCD), such as a CCD camera.
Preparation of Reaction Chamber and Flow Path
[0076] Borofloat glass wafers (Precision Glass & Optics, Santa
Ana, Calif.) are pre-etched for a short period in concentrated HF
(hydrofluoric acid) and cleaned before deposition of an amorphous
silicon sacrificial layer in a plasma-enhanced chemical vapor
deposition (PECVD) system (PEII-A, Technics West, San Jose,
Calif.). Wafers are primed with hexamethyldisilazane (HMDS),
spin-coated with photoresist (Shipley 1818, Marlborough, Mass.) and
soft-baked. A contact mask aligner (Quintel Corp., San Jose,
Calif.) is used to expose the photoresist layer with one or more
mask designs, and the exposed photoresist removed using a mixture
of Microposit developer concentrate (Shipley) and water. Developed
wafers are hard-baked and the exposed amorphous silicon removed
using CF.sub.4 (carbon tetrafluoride) plasma in a PECVD reactor.
Wafers are chemically etched with concentrated HF to produce the
reaction chamber 11, 220 and flow path 12. The remaining
photoresist is stripped and the amorphous silicon removed. Using
these methods, microchannels of about 50 to 100 .mu.m diameter may
be prepared. Smaller diameter channels may be prepared by known
methods, such as coating the inside of the microchannel to narrow
the diameter, or using nanolithography, focused electron beam,
focused ion beam or focused atom laser techniques.
[0077] Access holes are drilled into the etched wafers with a
diamond drill bit (Crystalite, Westerville, Ohio). A finished chip
is prepared by thermally bonding two complementary etched and
drilled plates to each other in a programmable vacuum furnace
(Centurion VPM, J. M. Ney, Yucaipa, Calif.). Alternative exemplary
methods for fabrication of a chip incorporating a reaction chamber
11, 220 and flow path 12 are disclosed in U.S. Pat. Nos. 5,867,266
and 6,214,246. In certain embodiments of the invention, a nylon
filter with a molecular weight cutoff of 2,500 daltons is inserted
between the reaction chamber 11, 220 and the flow path 12 to
prevent exonuclease 15 from leaving the reaction chamber 11,
220.
Nucleic Acid Preparation and Exonuclease Treatment
[0078] Human chromosomal DNA is purified according to Sambrook et
al. (1989). Following digestion with Bam H1, the genomic DNA
fragments are inserted into the multiple cloning site of the
pBluescript.RTM. II phagemid vector (Stratagene, Inc., La Jolla,
Calif.) and grown up in E. coli. After plating on
ampicillin-containing agarose plates a single colony is selected
and grown up for sequencing. Single-stranded DNA copies of the
genomic DNA insert are rescued by co-infection with helper phage.
After digestion in a solution of proteinase K:sodium dodecyl
sulphate (SDS), the DNA is phenol extracted and then precipitated
by addition of sodium acetate (pH 6.5, about 0.3 M) and 0.8 volumes
of 2-propanol. The DNA containing pellet is resuspended in
Tris-EDTA buffer and stored at -20.degree. C. until use. Agarose
gel electrophoresis shows a single band of purified DNA.
[0079] M13 forward primers complementary to the known
pBluescript.RTM. sequence, located next to the genomic DNA insert,
are purchased from Midland Certified Reagent Company (Midland,
Tex.). The primers are covalently modified to contain a biotin
moiety attached to the 5' end of the oligonucleotide. The biotin
group is covalently linked to the 5'-phosphate of the primer via a
(CH.sub.2).sub.6 spacer. Biotin-labeled primers are allowed to
hybridize to the ssDNA template molecules prepared from the
pBluescript.RTM. vector. The primer-template complexes are then
attached to streptavidin coated beads 14 according to Dorre et al.
(Bioimaging 5:139-152, 1997). At appropriate DNA dilutions, a
single primer-template complex is attached to a single bead 14. A
bead 14 containing a single primer-template complex is inserted
into the reaction chamber 11, 220 of a sequencing apparatus 10,
100, 210.
[0080] The primer-template is incubated with modified T7 DNA
polymerase (United States Biochemical Corp., Cleveland, Ohio). The
reaction mixture contains unlabeled deoxyadenosine-5'-triphosphate
(dATP) and deoxyguanosine-5'-triphosphate (dGTP),
digoxigenin-labeled deoxyuridine-5'-triphosphate (digoxigenin-dUTP)
and rhodamine-labeled deoxycytidine-5'-triphosphate
(rhodamine-dCTP). The polymerization reaction is allowed to proceed
for 2 hours at 37.degree. C. After synthesis of the digoxigenin and
rhodamine labeled nucleic acid 13, the template strand is separated
from the labeled nucleic acid 13, and the template strand, DNA
polymerase and unincorporated nucleotides are washed out of the
reaction chamber 11, 220.
[0081] Exonuclease 15 activity is initiated by addition of
exonuclease III 15 to the reaction chamber 11, 220. The reaction
mixture is maintained at pH 8.0 and 37.degree. C. As nucleotides
16, 130 are released from the 3' end 17 of the nucleic acid 13,
they are transported by microfluidic flow down the flow path 12
past the detection unit 18, 180, 300.
Detection of Labeled Nucleotides
[0082] The detection unit 18, 180, 300 comprises a laser 19, 320
and Raman detector 21, 310. The excitation beam 20, 330 is
generated by a titaniumsapphire laser 19, 320 (Tsunami by
Spectra-Physics) at a near-infrared wavelength (750-950 nm) or a
galium aluminum arsenide diode laser 19, 320 (PI-ECL series by
Process Instruments) at 785 nm or 830 nm. Pulsed laser beams 20,
330 or continuous beams 20, 330 can be used. The excitation beam
20, 330 is reflected by a dichroic mirror (holographic notch filter
by Kaiser Optical or an interference filter by Chroma or Omega
Optical) into a collinear geometry with the collected beam. The
reflected beam passes a microscope objective (Nikon LU series), and
is focused onto a micro-well, flow path (micro-channel) 12 or
flow-through cell 170, 290 where target nucleotides 16, 130 are
located. The Raman scattered light from the target nucleotides 16,
130 is collected by the same microscope objective, and passes the
dichroic mirror to the Raman detector 21, 310. The Raman detector
21, 310 comprises a focusing lens, a spectrograph, and an array
detector. The focusing lens focuses the Raman scattered light
through the entrance slit of the spectrograph. The spectrograph
(RoperScientific) comprises a grating that disperses the light by
its wavelength. The dispersed light is imaged onto an array
detector (back-illuminated deep-depletion CCD camera by
RoperScientific). The array detector is connected to a controller
circuit, which is connected to a computer for data transfer and
control of the detector 21, 310 function.
[0083] The Raman detector 21, 310 is capable of detecting and
identifying single nucleotides 16, 130 of dATP, dGTP,
rhodamine-dCTP and digoxigenin-dUTP moving past the detector 21,
310. Data on the time course for labeled nucleotide detection is
compiled and analyzed to obtain the sequence of the nucleic acid
13.
Example 2
Nucleic Acid Sequencing Using Covalent Attachment to
Nanoparticles
[0084] Another exemplary embodiment of the invention is disclosed
in FIG. 2. Nucleotides 16, 130 are released from a nucleic acid 13
by exonuclease 15 activity. In certain embodiments of the
invention, the nucleotides 16, 130 are unlabeled. Such embodiments
do not involve incorporation of labeled nucleotides into a
complementary strand 13 using primers and polymerases. Rather,
nucleic acids 13 directly purified from any organ, tissue and/or
cell sample or obtained by known cloning methods may be directly
sequenced. In some embodiments of the invention, a single molecule
of single-stranded RNA or DNA 13 may be attached to a surface 14
and treated with an exonuclease 15. Released nucleotides 16, 130
travel down a flow path 12. The flow path 12 may be contiguous with
or identical to a microfluidic channel 110, 160, 260, 280.
[0085] Nucleotides 16, 130 from the reaction chamber 11, 220 are
mixed with gold and/or silver nanoparticles 140. Silver
nanoparticles 140 are prepared according to Lee and Meisel (J.
Phys. Chem. 86:3391-3395, 1982). Gold nanoparticles 140 are
purchased from Polysciences, Inc. (Warrington, Pa.). Gold
nanoparticles 140 are available from Polysciences, Inc. in 5, 10,
15, 20, 40 and 60 nm sizes. In the present non-limiting Example, 60
nm gold nanoparticles 140 are used.
[0086] Prior to exposure to nucleotides 16, 130, surface-modified
nanoparticles 140 are coated with a silane, such as
3-glycidoxypropyltrimethoxysilane (GOP), a reactive linker
compound. GOP contains a terminal highly reactive epoxide group.
Nanoparticles 140 may be modified to contain hydroxyl groups to
allow covalent attachment of GOP. The silanized nanoparticles 140
are mixed with nucleotides 16, 130 and allowed to form covalent
cross-links with the nucleotides 16, 130. The
nucleotide-nanoparticle complexes 150 pass through a flow through
cell 170, 290 and are identified by SERS, SERRS and/or CARS using a
Raman detection unit 18, 180, 300. Because of the close proximity
of the nucleotides 16, 130 to the nanoparticles 140, the Raman
signals are greatly enhanced, allowing detection of single
nucleotides 16, 130 passing through the flow-through cell 170,
290.
Example 3
Apparatus for Nucleic Acid Sequencing
[0087] FIG. 3 shows another exemplary embodiment of the invention.
A DNA sequencing apparatus 10, 100, 210 comprises a reaction
chamber 11, 220 in fluid communication with an influx channel 230
and an efflux channel 240. Fluid movement may be controlled through
the use of one or more valves 250. A microfluidic channel 130, 260
is also in fluid communication with the reaction chamber 11, 220.
Nucleotides 16, 130 released from one or more nucleic acids 13 by
exonuclease 15 activity exit the reaction chamber 11, 220 through
the microfluidic channel 110, 260. The nucleotides 16, 130 are
mixed with nanoparticles 140 that move through a nanoparticle
channel 120, 270 in fluid communication with the microfluidic
channel 110, 260. Covalent attachment of nucleotides 16, 130 to
nanoparticles 140 occurs within an attachment channel 160, 280. The
covalently bound nucleotide-nanoparticle complexes 150 pass through
a flow-through cell 170, 290 where the nucleotides 16, 130 are
identified by a Raman detection unit 18, 180, 300. The detection
unit 18, 180, 300 comprises a laser 19, 320 and Raman detector 21,
310. The laser emits an excitation beam 20, 330 that excites
nucleotides 16, 130 within the flow-through cell 170, 290. Excited
nucleotides 16, 130 emit a Raman signal that is detected by the
Raman detector 21, 310.
[0088] In certain embodiments of the invention, nanoparticles 140
may be recovered in a recycling chamber 340. The nanoparticles are
chemically treated, for example with acid solutions, and then
washed to remove bound nucleotides 16, 130, linker compounds and
any other attached or adsorbed molecules. The nanoparticles 140 may
be recycled to a nanoparticle reservoir 370 via a recycling channel
360. In some embodiments of the invention, nanoparticles 140 may be
coated with a linker compound, such as GOP, in the recycling
channel 360 and/or the nanoparticle reservoir 370. Waste effluent
is removed from the recycling chamber 340 via a waste channel
350.
[0089] All of the METHODS and APPARATUS disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. It will be apparent to those of
skill in the art that variations may be applied to the METHODS and
APPARATUS described herein without departing from the concept,
spirit and scope of the claimed subject matter. More specifically,
it will be apparent that certain agents that are both chemically
and physiologically related may be substituted for the agents
described herein while the same or similar results would be
achieved. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the claimed subject matter.
* * * * *
References