U.S. patent application number 11/753361 was filed with the patent office on 2010-10-21 for methods to increase nucleotide signals by raman scattering.
This patent application is currently assigned to INTEL CORPORATION. Invention is credited to Andrew Arthur Berlin, Selena Chan, Steven J. Kirch, Tae-Woong Koo, Gabi Neubauer, Valluri Rao, Xing Su, Narayan Sundararajan, Mineo Yamakawa.
Application Number | 20100267013 11/753361 |
Document ID | / |
Family ID | 34435316 |
Filed Date | 2010-10-21 |
United States Patent
Application |
20100267013 |
Kind Code |
A1 |
Su; Xing ; et al. |
October 21, 2010 |
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. In other
embodiments, unlabeled nucleic acids are used. Exonuclease
treatment of the nucleic acid results in the release of labeled or
unlabeled nucleotides that are detected by Raman spectroscopy. In
alternative embodiments of the invention, nucleotides released from
a nucleic acid by exonuclease treatment are covalently cross-linked
to nanoparticles and detected by surface enhanced Raman
spectroscopy (SERS), surface enhanced resonance Raman spectroscopy
(SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS).
Other embodiments of the invention concern apparatus for nucleic
acid sequencing.
Inventors: |
Su; Xing; (Cupertino,
CA) ; Berlin; Andrew Arthur; (San Jose, CA) ;
Chan; Selena; (San Jose, CA) ; Kirch; Steven J.;
(Pleasanton, CA) ; Koo; Tae-Woong; (Cupertino,
CA) ; Neubauer; Gabi; (Los Gatos, CA) ; Rao;
Valluri; (Saratoga, CA) ; Sundararajan; Narayan;
(San Francisco, CA) ; Yamakawa; Mineo; (Campbell,
CA) |
Correspondence
Address: |
Pillsbury Winthrop Shaw Pittman LLP;(INTEL)
P.O. Box 10500
McLean
VA
22102
US
|
Assignee: |
INTEL CORPORATION
Santa Clara
CA
|
Family ID: |
34435316 |
Appl. No.: |
11/753361 |
Filed: |
May 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10660902 |
Sep 12, 2003 |
7238477 |
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11753361 |
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10099287 |
Mar 14, 2002 |
6972173 |
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10660902 |
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09962555 |
Sep 24, 2001 |
6982165 |
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10099287 |
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Current U.S.
Class: |
435/6.12 ;
435/288.7; 977/773 |
Current CPC
Class: |
C12Q 2563/155 20130101;
C12Q 2565/632 20130101; C12Q 2521/319 20130101; C12Q 2565/632
20130101; C12Q 2565/632 20130101; C12Q 1/6869 20130101; C12Q 1/6869
20130101; C12Q 1/6869 20130101; C12Q 1/6869 20130101 |
Class at
Publication: |
435/6 ;
435/288.7; 977/773 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Claims
1. A method comprising: a) using an exonuclease to release
unlabeled nucleotides from one end of one or more nucleic acid
molecules; b) separating the unlabeled nucleotides from the
exonuclease and the one or more nucleic acid molecules; c)
identifying the unlabeled nucleotides by Raman spectroscopy to
determine identified nucleotides; and d) determining the sequence
of the nucleic acid from the identified nucleotides.
2. The method of claim 1, wherein single molecules of the unlabeled
nucleotides are identified by Raman spectroscopy.
3. The method of claim 2, wherein a single nucleic acid molecule is
sequenced.
4. The method of claim 1, wherein multiple nucleic acid molecules
are sequenced simultaneously.
5. The method of claim 1, wherein the one or more nucleic acid
molecules is attached to a surface.
6. The method of claim 1, wherein the unlabeled nucleotides are
identified by surface enhanced Raman spectroscopy (SERS), surface
enhanced resonance Raman spectroscopy (SERRS) and/or coherent
anti-Stokes Raman spectroscopy (CARS).
7-13. (canceled)
14. The method of claim 1, wherein the unlabeled nucleotides are
attached to nanoparticles after the nucleotides are removed from
the nucleic acid.
15. The method of claim 1, wherein the unlabeled nucleotides are
attached to nanoparticles before the nucleotides are removed from
the nucleic acid.
16. The method of claim 15, wherein nanoparticles are attached to
the 3' end of the nucleic acid.
17-21. (canceled)
22. A method comprising: a) obtaining nucleotides that are attached
to Raman labels; b) synthesizing a nucleic acid comprising labeled
nucleotides; c) removing nucleotides from one end of the nucleic
acid; d) identifying the nucleotides by Raman spectroscopy; and e)
determining the sequence of the nucleic acid.
23. The method of claim 22, wherein single nucleotide molecules are
identified by Raman spectroscopy
24. The method of claim 22, wherein each type of nucleotide is
labeled with a distinguishable Raman label.
25. The method of claim 22, wherein only pyrimidine nucleotides are
labeled with Raman labels.
26. The method of claim 22, further comprising: (i) obtaining at
least one template nucleic acid molecule; (ii) hybridizing the
template nucleic acid molecule to a primer; and (iii) adding a DNA
polymerase to synthesize said nucleic acid.
27. An apparatus comprising: a) a reaction chamber; b) a
microfluidic channel in fluid communication with the reaction
chamber; c) a flow-through cell in fluid communication with the
microfluidic channel; and d) a Raman detection unit operably
coupled to the flow-through cell.
28. The apparatus of claim 27, wherein the Raman detector is
capable of detecting single molecules of nucleotides.
29. The apparatus of claim 28, wherein the nucleotides are
unlabeled.
30. The apparatus of claim 27, further comprising nanoparticles in
the flow-through cell.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
patent application Ser. No. 10/099,287, filed on Mar. 14, 2002; and
a continuation-in-part of U.S. patent application Ser. No.
09/962,555, filed Sep. 24, 2001.
FIELD OF THE INVENTION
[0002] 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
[0003] 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.
[0004] 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.
[0005] 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. It also typically requires the use of fluorescent
or radioactive labels, which can potentially pose safety and waste
disposal problems.
[0006] More recently, methods for nucleic acid sequencing have been
developed involving hybridization to short oligonucleotides of
defined sequenced, attached to specific locations on DNA chips.
Such methods may be used to infer short nucleic acid sequences or
to detect the presence of a specific nucleic acid in a sample, but
are not suited for identifying long nucleic acid sequences.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] 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.
[0008] 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.
[0009] 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 enhanced Raman spectroscopy (SERS) 180.
[0010] FIG. 3 illustrates another exemplary apparatus 210 (not to
scale) for nucleic acid 13 sequencing.
[0011] FIG. 4 shows the Raman spectra of all four deoxynucleoside
monophosphates (dNTPs) at 100 mM concentration, using a 100
millisecond data collection time. Characteristic Raman emission
peaks for as shown for each different type of nucleotide. The data
were collected without surface-enhancement or labeling of the
nucleotides.
[0012] FIG. 5 shows SERS detection of 1 nM guanine, obtained from
dGMP by acid treatment according to Nucleic Acid Chemistry, Part 1,
L. B. Townsend and R. S. Tipson (Eds.), Wiley-Interscience, New
York, 1978.
[0013] FIG. 6 shows SERS detection of 10 nM cytosine, obtained from
dCMP by acid treatment.
[0014] FIG. 7 shows SERS detection of 100 nM thymine, obtained from
dTMP by acid treatment.
[0015] FIG. 8 shows SERS detection of 100 pM adenine, obtained from
dAMP by acid treatment.
[0016] FIG. 9 shows a comparative SERS spectrum of a 500 nM
solution of deoxyadenosine triphosphate covalently labeled with
fluorescein (upper trace) and unlabeled dATP (lower trace). The
dATP-fluorescein was obtained from Roche Applied Science
(Indianapolis, Ind.). A strong increase in the SERS signal was
detected in the fluorescein labeled dATP.
[0017] FIG. 10 shows the SERS detection of a 0.9 nM (nanomolar)
solution of adenine. The detection volume was 100 to 150
femtoliters, containing an estimated 60 molecules of adenine.
[0018] FIG. 11 shows the SERS detection of a rolling circle
amplification product, using a single-stranded, circular M13 DNA
template.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0019] The disclosed methods and apparatus are of use for the
rapid, automated sequencing of nucleic acids. In particular
embodiments of the invention, the methods and apparatus are
suitable for obtaining the sequences of very long nucleic acid
molecules 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 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.
[0020] 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. In other
embodiments of the invention, multiple copies of a nucleic acid
molecule 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 and its complementary strand may be sequenced to confirm
the accuracy of the sequence information. In various embodiments, a
nucleic acid to be sequenced may be attached, either covalently or
non-covalently to a surface. In particular embodiments, nucleotides
may be released from a surface-attached nucleic acid, for example
by exonuclease treatment. Released nucleotides may be transported,
for example, through a microfluidic system to a Raman detector, to
allow detection of released nucleotides without background Raman
signals from the nucleic acid, exonuclease and/or other components
of the system.
[0021] In certain embodiments of the invention, the nucleic acid to
be sequenced is DNA, although it is contemplated that other nucleic
acids 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.
[0022] In various embodiments of the invention, unlabeled
nucleotides may be detected by Raman spectroscopy, for example by
surface enhanced Raman spectroscopy (SERS), surface enhanced
resonance Raman spectroscopy (SERBS), coherent anti-Stokes Raman
spectroscopy (CARS) or other known Raman detection techniques. In
alternative embodiments, nucleotides may be covalently attached to
Raman labels to enhance the Raman signal. In some embodiments,
labeled nucleotides may be incorporated into a newly synthesized
nucleic acid strand 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 complementary in sequence to the
template. The labeled strand may be separated from the unlabeled
template, for example by heating to about 95.degree. C. or other
known methods. The two strands may be separated from each other by
techniques well known in the art. For example, the primer
oligonucleotide may be covalently modified with a biotin residue
and the resulting biotinylated nucleic acid may be separated by
binding to an avidin or streptavidin coated surface.
[0023] In alternative embodiments of the invention, either labeled
or unlabeled single-stranded nucleic acid molecules may be digested
with one or more exonucleases. The skilled artisan will realize
that the disclosed methods are not limited to exonucleases per se,
but may utilize any enzyme or other reagent capable of sequentially
removing nucleotides from at least one end of a nucleic acid. In
certain embodiments of the invention, labeled or unlabeled
nucleotides are sequentially released from the 3' end of the
nucleic acid. After separation from the nucleic acid, the
nucleotides are detected by a Raman detection unit. Information on
sequentially detected nucleotides is used to compile a sequence of
the nucleic acid. Nucleotides released from the 3' end of a nucleic
acid may be transported down a microfluidic flow path past a Raman
detector. In particular embodiments, the detector is capable of
detecting labeled or unlabeled nucleotides at the single molecule
level. The order of detection of the nucleotides by the Raman
detector is the same as the order in which the nucleotides are
released from the 3' end of the nucleic acid. The sequence of the
nucleic acid can thus be determined by the order in which released
nucleotides are detected. Where a complementary strand is sequence,
the template strand will be complementary in sequence according to
standard Watson-Crick hydrogen bond base-pairing (i.e., A-T and G-C
or A-U and G-C, depending on whether DNA or RNA is sequenced).
[0024] In certain alternative embodiments, a tag molecule may be
added to a reaction chamber or flow path upstream of the detection
unit. The tag molecule binds to and tags free nucleotides as they
are released from the nucleic acid molecule. This post-release
tagging avoids problems that are encountered when the nucleotides
of the nucleic acid molecule are tagged before their release into
solution. For example, the use of bulky Raman label molecules may
provide steric hindrance when each nucleotide incorporated into a
nucleic acid molecule is labeled before exonuclease treatment,
reducing the efficiency and increasing the time required for the
sequencing reaction.
[0025] 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). In alternative embodiments, no nucleotides are
labeled and the unlabeled nucleotides are identified by Raman
spectroscopy.
[0026] In specific embodiments of the invention, the Raman signals
may be enhanced by covalent attachment of nucleotides to
nanoparticles. Nanoparticles may be prepared as discussed below and
activated by attachment of highly reactive groups, for example
epoxide groups, using known methods. For example, nanoparticles may
be coated with 3-glycidoxypropyltrimethoxysilane (GOP). GOP
contains a terminal highly reactive epoxide group. The use of
highly reactive groups such as epoxides allows for rapid formation
of covalent bonds between nanoparticles and nucleotides. In some
embodiments, the nucleotides may be released from a nucleic acid by
exonuclease activity and then reacted with nanoparticles to allow
covalent bond formation. Various methods may be employed to allow
sufficient time for covalent bonds to form before the
nanoparticle-nucleotide complex. For example, a solution containing
released nucleotides and activated nanoparticles may be transported
by microfluidic flow down a relatively long flow path, allowing
sufficient time for covalent bond formation to occur before the
nucleotide-nanoparticle complex passes in front of the Raman
detector. Flow rate may be further decreased by use of a fluid of
high viscosity, for example a glycerol solution. Methods of
microfluidic flow of high viscosity solutions are known in the
art.
[0027] Alternatively, a cyclic process may be employed, wherein a
nanoparticle is first allowed to bind to the 3' end of a nucleic
acid. The nanoparticle and attached nucleotide may be released from
the 3' end of the nucleic acid by exonuclease activity or chemical
treatment. For example, the phosphodiester bond attaching the
terminal nucleotide to the nucleic acid may be cleaved by treatment
with acid or base. The electron-withdrawing effect of the attached
nanoparticle may render the terminal phosphodiester bond
particularly labile to cleavage, allowing removal of a single
nucleotide at a time. Following release, another nanoparticle may
be reacted with the 3' end of the nucleic acid and the process
repeated in a cycle. Alternatively, a 3' exonuclease may be used to
release the nucleotide-nanoparticle complex. Steric hindrance from
the nanoparticle with exonuclease activity may be avoided by using
a linker arm to attach the reactive group (e.g., epoxide) to the
nanoparticle. Attachment of the nanoparticle before release of the
terminal nucleotide would allow ample time for covalent bond
formation. In other alternative embodiments of the invention, the
rate of exonuclease activity may be adjusted to coordinate the
rates of release of nucleotides and their covalent attachment to
nanoparticles. For example, a reaction chamber containing a nucleic
acid and exonuclease may be temperature controlled to a reduced
temperature, of between 0.degree. C. and room temperature. Once an
appropriate temperature has been determined, the nucleotides
released by exonuclease activity may enter a flow path where they
are mixed with reactive nanoparticles at an elevated temperature,
between room temperature and 100.degree. C. The elevated
temperature would increase the rate of reactivity of nucleotide and
nanoparticle. In various embodiments, temperature ranges from about
0.degree. C. to 5.degree. C., 5.degree. C. to 10.degree. C.,
10.degree. C. to 15.degree. C., 15.degree. C. to 20.degree. C. or
20.degree. C. to 25.degree. C. may be used to regulate exonuclease
activity. In certain embodiments, temperature ranges from about
20.degree. C. to 25.degree. C., 25.degree. C. to 30.degree. C.,
30.degree. C. to 35.degree. C., 35.degree. C. to 40.degree. C.,
40.degree. C. to 45.degree. C., 45.degree. C. to 50.degree. C.,
50.degree. C. to 55.degree. C., 55.degree. C. to 60.degree. C.,
60.degree. C. to 65.degree. C., 65.degree. C. to 70.degree. C.,
70.degree. C. to 75.degree. C., 75.degree. C. to 80.degree. C. or
80.degree. C. to 95.degree. C. may be used to regulate the rate of
covalent bond formation between nanoparticle and nucleotide. It is
well within the routine skill in the art to assay reaction rates as
a function of temperature and to select appropriate temperature
ranges to coordinate the rates of exonuclease activity and
nucleotide-nanoparticle cross-linking.
[0028] In some embodiments of the invention, the nanoparticles are
silver or gold, but other types of nanoparticles known to provide
surface enhanced Raman signals are contemplated. The nanoparticles
may either be single nanoparticles, aggregates of nanoparticles, or
some mixture of single and aggregated nanoparticles. In certain
embodiments, a linker compound may be used to attach the
nucleotides to the nanoparticles. 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, 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, two or more nanoparticles may be attached together
using linker compounds.
[0029] The nanoparticle-nucleotide complexes may pass through a
flow-through cell where they are detected by SERS, SERRS and/or
CARS using a Raman detection unit. In some alternative embodiments
of the invention, the nucleotides may be unmodified, while in other
alternative embodiments the nucleotides may be modified with one or
more Raman labels. In certain embodiments, each type of nucleotide
may be attached to a distinguishable Raman label. In other
embodiments only pyrimidines may be labeled.
DEFINITIONS
[0030] As used herein, "a" or "an" may mean one or more than one of
an item.
[0031] As used herein, "operably coupled" means that there is a
functional interaction between two or more units. For example, a
detector may be "operably coupled" to a flow-through cell if the
detector is arranged so that it may detect analytes, such as
nucleotides, as they pass through the flow-through cell.
[0032] "Nucleic acid" encompasses DNA, RNA, single-stranded,
double-stranded or triple stranded and any chemical modifications
thereof. Virtually any modification of the nucleic acid is
contemplated. As used herein, a single stranded nucleic acid may be
denoted by the prefix "ss", a double stranded nucleic acid by the
prefix "ds", and a triple stranded nucleic acid by the prefix "ts."
A "nucleic acid" 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.
[0033] A "nucleoside" 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.
[0034] A "nucleotide" refers to a nucleoside further comprising at
least one phosphate group covalently attached to the pentose sugar.
In some embodiments of the invention, the nucleotides are
ribonucleoside monophosphates or deoxyribonucleoside
monophosphates, although it is anticipated that nucleoside
diphosphates or triphosphates could be produced and detected. In
other embodiments of the invention, nucleosides may be released
from the nucleic acid molecule. It is contemplated that various
substitutions or modifications may be made in the structure of the
nucleotides, so long as they are capable of being incorporated into
a nucleic acid by polymerase activity and released by an
exonuclease or equivalent reagent. In embodiments of the invention
involving one or more labels attached to one or more types of
nucleotide, the label may be attached to any portion of the
nucleotide, such as the base, the sugar or the phosphate groups or
their analogs. 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.
[0035] 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 C.sub.60, 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
[0036] Nucleic acid molecules to be sequenced may be prepared by
any technique known in the art. In certain embodiments of the
invention, the nucleic acids are naturally occurring DNA or RNA
molecules. Virtually any naturally occurring nucleic acid 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 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) is to be
sequenced, an ssDNA 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 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. As discussed above,
one of the two strands of double-stranded DNA may be separated, for
example, by biotin labeling and attachment to avidin or
streptavidin using known techniques.
[0037] Although certain embodiments of the invention concern
preparation of naturally occurring nucleic acids, virtually any
type of nucleic acid that can serve as a substrate for an
exonuclease or equivalent reagent could potentially be sequenced.
For example, nucleic acids prepared by various amplification
techniques, such as polymerase chain reaction (PCR.TM.)
amplification, could be sequenced. (See U.S. Pat. Nos. 4,683,195,
4,683,202 and 4,800,159.) Nucleic acids 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 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 are well known.
Isolation of Single Nucleic Acid Molecules
[0038] In certain embodiments of the invention, the nucleic acid
molecule to be sequenced is a single molecule of ssDNA or ssRNA. A
variety of methods for selection and manipulation of single nucleic
acid molecules 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.)
[0039] In certain embodiments of the invention, microfluidics or
nanofluidics may be used to sort and isolate nucleic acid
molecules. Hydrodynamics may be used to manipulate the movement of
nucleic acids into a microchannel, microcapillary, or a micropore.
In one embodiment of the invention, hydrodynamic forces may be used
to move nucleic acid molecules across a comb structure to separate
single nucleic acid molecules. Once the nucleic acid molecules have
been separated, hydrodynamic focusing may be used to position the
molecules within a reaction chamber. A thermal or electric
potential, pressure or vacuum can also be used to provide a motive
force for manipulation of nucleic acids. In exemplary embodiments
of the invention, manipulation of nucleic acids 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).
[0040] In another embodiment of the invention, a sample containing
the nucleic acid molecule may be diluted prior to coupling to an
immobilization surface. In exemplary embodiments of the invention,
the immobilization surface may be in the form of magnetic or
non-magnetic beads or other discrete structural units. At an
appropriate dilution, each bead will have a statistical probability
of binding zero or one nucleic acid molecule. Beads with one
attached nucleic acid molecule 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 and the nucleic acids, it may be possible to use a magnetic
filter and mass separation to separate beads containing a single
bound nucleic acid molecule. In other embodiments of the invention,
multiple nucleic acids attached to a single bead or other
immobilization surface may be sequenced.
[0041] In alternative embodiments of the invention, a coated fiber
tip may be used to generate single molecule nucleic acids for
sequencing (e.g., U.S. Pat. No. 6,225,068). In other alternative
embodiments, the immobilization surfaces may be prepared to contain
a single molecule of avidin or other cross-linking agent. Such a
surface could attach a single biotinylated nucleic acid molecule to
be sequenced. This embodiment is not limited to the avidin-biotin
binding system, but may be adapted to any known coupling
system.
[0042] In other alternative embodiments of the invention, an
optical trap may be used for manipulation of single molecule
nucleic acid molecules 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
[0043] Certain embodiments of the invention may involve attaching a
label to the nucleotides to facilitate their measurement by the
detection unit. 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',T-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.).
[0044] 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.
[0045] Labels may be attached directly to the nucleotides 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, are commercially available (e.g., Molecular
Probes, Eugene, Oreg.). Methods for preparing labeled nucleotides
and incorporating them into nucleic acids are known (e.g., U.S.
Pat. Nos. 4,962,037; 5,405,747; 6,136,543; 6,210,896).
Nanoparticles
[0046] Certain embodiments of the invention involve the use of
nanoparticles to enhance the Raman signal obtained from
nucleotides. In some embodiments of the invention, the
nanoparticles are silver or gold nanoparticles, although any
nanoparticles capable of providing a surface enhanced Raman
spectroscopy (SERS) signal may be used. In alternative embodiments
of the invention, the nanoparticles may be nanoprisms (Jin et al.,
Science 294:1902-3, 2001.) In various embodiments of the invention,
nanoparticles of between 1 nm and 2 micrometers (.mu.m) in diameter
may be used. In alternative embodiments of the invention,
nanoparticles of between 2 nm to 1 .mu.m, 5 nm to 500 nm, 5 nm to
200 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 with an average
diameter of 10 to 50 nm, 50 to 100 nm or about 100 nm are
contemplated. The nanoparticles may be approximately spherical,
rod-like, edgy, faceted or pointy in shape, although nanoparticles
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.).
[0047] In certain embodiments of the invention, the nanoparticles
may be single nanoparticles and/or random aggregates of
nanoparticles (colloidal nanoparticles). In other embodiments of
the invention, nanoparticles may be cross-linked to produce
particular aggregates of nanoparticles, such as dimers, trimers,
tetramers or other aggregates. Certain alternative embodiments may
use heterogeneous mixtures of aggregates of different size, while
other alternative embodiments may use homogenous populations of
nanoparticles. In certain embodiments, aggregates containing a
selected number of nanoparticles (dimers, trimers, etc.) may be
enriched or purified by known techniques, such as
ultracentrifugation in sucrose solutions. In various embodiments of
the invention, nanoparticle aggregates of about 100, 200, 300, 400,
500, 600, 700, 800, 900 to 1000 nm in size or larger are
contemplated.
[0048] Methods of cross-linking nanoparticles are known (e.g.,
Feldheim, "Assembly of metal nanoparticle arrays using molecular
bridges," The Electrochemical Society Interface, Fall, 2001, pp.
22-25). Gold nanoparticles may be cross-linked, for example, using
bifunctional linker compounds bearing terminal thiol or sulfhydryl
groups. Upon reaction with gold nanoparticles, the linker forms
nanoparticle 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 (Feldheim, 2001). The use of an excess of
nanoparticles to linker compounds prevents formation of multiple
cross-links and nanoparticle precipitation. Aggregates of silver
nanoparticles may be formed by standard synthesis methods known in
the art.
[0049] In alternative embodiments of the invention, the
nanoparticles may be modified to contain various reactive groups
before they are attached to linker compounds. Modified
nanoparticles are commercially available, such as Nanogold.RTM.
nanoparticles from Nanoprobes, Inc. (Yaphank, N.Y.). Nanogold.RTM.
nanoparticles may be obtained with either single or multiple
maleimide, amine or other groups attached per nanoparticle. The
Nanogold.RTM. nanoparticles are also available in either positively
or negatively charged form. Such modified nanoparticles may be
attached to a variety of known linker compounds to provide dimers,
trimers or other aggregates of nanoparticles.
[0050] The type of linker compound used is not limiting, so long as
it results in the production of small aggregates of nanoparticles
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.
[0051] In various embodiments of the invention, the nanoparticles
may be covalently attached to nucleotides. In alternative
embodiments of the invention, the nucleotides may be directly
attached to the nanoparticles, or may be attached to linker
compounds that are covalently or non-covalently bonded to the
nanoparticles. In such embodiments of the invention, rather than
cross-linking two or more nanoparticles together the linker
compounds may be used to attach a nucleotide to a nanoparticle or a
nanoparticle aggregate. In particular embodiments of the invention,
the nanoparticles may be coated with derivatized silanes. Such
modified silanes may be covalently attached to nucleotides using
standard methods. Various methods known for cross-linking nucleic
acids to surfaces discussed below may also be used to attach
nucleotides to nanoparticles. It is contemplated that the linker
compounds used to attach nucleotides 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.
[0052] In other embodiments of the invention, nucleotides may be
adsorbed on the surface of the nanoparticles or may be in close
proximity to the nanoparticles (between about 0.2 and 1.0 nm). The
skilled artisan will realize that it covalent attachment of the
nucleotides to nanoparticles is not required in order to generate
an enhanced Raman signal by SERS, SERRS or CARS.
[0053] In some embodiments of the invention, the nucleotides may be
attached to nanoparticles as they travel down a microfluidic
channel to form nucleotide-nanoparticle complexes. In certain
embodiments of the invention, the length of time available for the
cross-linking reaction to occur may be 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, the reactive groups may be
selected so that they can only attach the nanoparticles to
nucleotides, rather than cross-linking the nanoparticles to each
other. The selection and preparation of reactive cross-linking
groups capable of binding to nucleotides is known in the art. In
alternative embodiments of the invention, nucleotides may
themselves be covalently modified, for example with a sulfhydryl
group that can attach to gold nanoparticles.
[0054] In certain embodiments of the invention, nanoparticles may
be manipulated into microfluidic channels by any method known in
the art, such as microfluidics, nanofluidics, hydrodynamic focusing
or electro-osmosis. In some embodiments, use of charged linker
compounds or charged nanoparticles may facilitate manipulation of
nanoparticles through the use of electrical gradients.
Immobilization of Nucleic Acids
[0055] In certain embodiments of the invention, one or more nucleic
acid molecules may be attached to a surface 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 (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.
[0056] 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 and
exonuclease 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 to
surfaces are known in the art and may be employed. In certain
embodiments of the invention, the nucleic acid molecule is fixed in
place and immersed in a microfluidic flow down a flow path and/or
microfluidic channel that transports the released nucleotides past
a detection unit. In non-limiting examples, the microfluidic flow
may result from a bulk flow of solvent down a flow path and/or
microfluidic channel.
[0057] 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) move down a flow
path and/or microfluidic channel in response to an externally
applied electrical field.
[0058] Immobilization of nucleic acid molecules may be achieved by
a variety of known methods. In an exemplary embodiment of the
invention, immobilization may be achieved by coating a surface with
streptavidin or avidin and the subsequent attachment of a
biotinylated nucleic acid (Holmstrom et al., Anal. Biochem.
209:278-283, 1993). Immobilization may also occur by coating a
silicon, glass or other surface with poly-L-Lys (lysine) or poly
L-Lys, Phe (phenylalanine), followed by covalent attachment of
either amino- or sulfhydryl-modified nucleic acids 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 through the use of
aminosilane.
[0059] Immobilization may take place by direct covalent attachment
of 5'-phosphorylated nucleic acids to chemically modified surfaces
(Rasmussen et al., Anal. Biochem. 198:138-142, 1991). The covalent
bond between the nucleic acid and the surface may be formed by
condensation with a water-soluble carbodiimide. This method
facilitates a predominantly 5-attachment of the nucleic acids via
their 5'-phosphates.
[0060] DNA is commonly bound to glass by first silanizing the glass
surface, then activating with carbodiimide or glutaraldehyde.
Alternative procedures may use reagents such as
3-glycidoxypropyltrimethoxysilane (GOP) or
aminopropyltrimethoxysilane (APTS) with DNA linked via amino
linkers incorporated at either the 3' or 5' end of the molecule.
DNA may be bound directly to membrane surfaces using ultraviolet
radiation. Other non-limiting examples of immobilization techniques
for nucleic acids are disclosed in U.S. Pat. Nos. 5,610,287,
5,776,674 and 6,225,068.
[0061] Bifunctional cross-linking reagents may be of use in various
embodiments of the invention, such as attaching a nucleic acid
molecule to a surface. 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 5,401,511. Cross-linking reagents include
glutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycol
diglycidyl ether (EGDE), and carbodiimides, such as
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
Nucleic Acid Synthesis
[0062] Polymerases
[0063] 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
Thermatoga maritima DNA polymerase, AmplitaqFS.TM. DNA polymerase,
Taquenase.TM. DNA polymerase, ThermoSequenase.TM., Taq DNA
polymerase, Qbeta.TM. replicase, T4 DNA polymerase, Thermus
thermophilus DNA polymerase, RNA-dependent RNA polymerase and SP6
RNA polymerase.
[0064] A number of polymerases are commercially available,
including Pwo DNA Polymerase (Boehringer Mannheim Biochemicals,
Indianapolis, Ind.); Bst Polymerase (Bio-Rad Laboratories,
Hercules, Calif.); IsoTherm.TM. 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 from labeled nucleotides are known (e.g.,
U.S. Pat. Nos. 4,962,037; 5,405,747; 6,136,543; 6,210,896).
Primers
[0065] 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.
Methods for synthesis of primers of any sequence are known. Other
embodiments of the invention involve sequencing a nucleic acid 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.
Nucleic Acid Digestion
[0066] In certain embodiments of the invention, methods of nucleic
acid sequencing involve binding of an exonuclease or equivalent
reagent to the free end of a nucleic acid molecule and removal of
nucleotides one at a time. Non-limiting examples of nucleic acid
digesting enzymes 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 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.).
[0067] The skilled artisan will realize that enzymes with
exonuclease activity may remove nucleotides from the 5' end, the 3'
end, or either end of nucleic acid molecules. They can show
specificity for RNA, DNA or both RNA and DNA. Their activity may
depend on the use of either single or double-stranded nucleic
acids. They may be differentially affected by salt concentration,
temperature, pH, or divalent cations. These and other properties of
exonucleases are known in the art. In certain embodiments of the
invention, the rate of exonuclease activity may be manipulated to
coincide with the optimal rate of analysis of nucleotides by the
detection unit. Various methods are known for adjusting the rate of
exonuclease activity, including adjusting the temperature,
pressure, pH, salt or divalent cation concentration in a reaction
chamber.
[0068] Although nucleoside monophosphates will generally be
released from nucleic acids by exonuclease activity, the
embodiments of the invention are not limited to detection of any
particular form of free nucleotide or nucleoside but encompass any
monomer that may be released from a nucleic acid.
Reaction Chamber and Integrated Chip
[0069] Some embodiments of the invention concern apparatus
comprising a reaction chamber designed to contain an immobilization
surface, nucleic acid molecule, exonuclease and nucleotides in an
aqueous environment. In some embodiments of the invention, the
reaction chamber 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.)
[0070] In certain embodiments of the invention, the reaction
chamber and any associated fluid channels, for example, a flow
path, microfluidic channels or channels to provide connections to
waste ports, to a nucleic acid loading port, to a nanoparticle
reservoir, to a source of exonuclease 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 and other components of the apparatus, such as the flow
path and/or microfluidic channels, 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.).
[0071] To facilitate detection of nucleotides by the detection unit
the material comprising the flow path or flow-through cell may be
selected to be transparent to electromagnetic radiation at the
excitation and emission frequencies used for the detection unit.
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 or flow-through cell that are opposite the detection unit may
be coated with silver, gold, platinum, copper, aluminum or other
materials that are relatively opaque to the detection unit. 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. Alternatively, the flow path or
flow-through cell may contain a mesh comprising silver, gold,
platinum, copper, aluminum or other Raman signal enhancing metal.
In other alternative embodiments of the invention, the flow path or
flow-through cell may contain metal nanoparticles.
Flow Path and Microfluidic Channels
[0072] In certain embodiments of the invention, the nucleotides
released from a nucleic acid are moved down a flow path and/or
microfluidic channels past a detection unit. A non-limiting example
of techniques for transport of nucleotides includes microfluidic
techniques. The flow path and/or microfluidic channels 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.). A microchannel flow path
may be from about 5 to 200 .mu.m in diameter, with a diameter of
about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200
.mu.m.
[0073] In certain embodiments of the invention, the nucleotides to
be detected move down the flow path and/or microfluidic channels by
bulk flow of solvent. In other embodiments of the invention,
microcapillary electrophoresis may be used to transport nucleotides
down the flow path and/or microfluidic channels. 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, occurs in response to an
imposed electrical field, negative on the reaction chamber side of
the apparatus and positive on the detection unit 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 that are
sequentially released from a nucleic acid. Because the purine
nucleotides (A, G) are larger than the pyrimidine nucleotides (C,
T, U) and would therefore migrate more slowly, the length of the
flow path and/or microfluidic channels and the corresponding
transit time past the detection unit may kept to a minimum to
prevent differential migration from mixing up the order of
nucleotides released from the nucleic acid. Alternatively, the
medium filling the microcapillary may be selected so that the
migration rates of purine and pyrimidine nucleotides down the flow
path and/or microfluidic channels 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).
[0074] In certain embodiments of the invention, flow paths and/or
microfluidic channels 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
to nanoparticles.
[0075] 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, using techniques known in the art.
Detection Unit
[0076] In various embodiments of the invention, the detection unit
is designed to detect and quantify nucleotides by Raman
spectroscopy. Methods for detection of micromolar concentrations of
nucleotides by Raman spectroscopy are known in the art. (See, e.g.,
U.S. Pat. Nos. 5,306,403; 6,002,471; 6,174,677). Exemplary methods
for detection of nucleotides at the single molecule level are
disclosed in the Examples below. 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 10.sup.6 or more for molecules adjacent to
roughened metal surfaces, such as silver, gold, platinum, copper or
aluminum surfaces.
[0077] A non-limiting example of a Raman detection unit is
disclosed in U.S. Pat. No. 6,002,471. An excitation beam is
generated by either a frequency doubled Nd:YAG laser at 532 nm
wavelength or a frequency doubled Ti:sapphire laser at 365 nm
wavelength. Pulsed laser beams or continuous laser beams may be
used. The excitation beam passes through confocal optics and a
microscope objective, and is focused onto the flow path and/or the
flow-through cell. The Raman emission light from the nucleotides 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, comprising an avalanche photodiode interfaced with
a computer for counting and digitization of the signal.
[0078] Another example of a Raman detection unit is disclosed in
U.S. Pat. No. 5,306,403, including a Spex Model 1403 double-grating
spectrophotometer with a gallium-arsenide photomultiplier tube (RCA
Model C31034 or Burle Industries Model C3103402) operated in the
single-photon counting mode. The excitation source comprises a
514.5 nm line argon-ion laser from SpectraPhysics, Model 166, and a
647.1 nm line of a krypton-ion laser (Innova 70, Coherent).
[0079] Alternative excitation sources include a nitrogen laser
(Laser Science Inc.) at 337 nm and a helium-cadmium laser (Liconox)
at 325 nm (U.S. Pat. No. 6,174,677), a light emitting diode, an
Nd:YLF laser, and/or various ions lasers and/or dye lasers. The
excitation beam may be spectrally purified with a bandpass filter
(Corion) and may be focused on the flow path and/or flow-through
cell using a 6.times. objective lens (Newport, Model L6X). The
objective lens may be used to both excite the nucleotides 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 and the emitted Raman
signal. A holographic notch filter (Kaiser Optical Systems, Inc.)
may be used to reduce Rayleigh scattered radiation. Alternative
Raman detectors 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 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.
[0080] Any suitable form or configuration of Raman spectroscopy or
related techniques known in the art may be used for detection of
nucleotides, 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
[0081] In certain embodiments of the invention, the nucleic acid
sequencing apparatus may comprise an information processing system.
The disclosed methods and apparatus 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. DI
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.
[0082] In particular embodiments of the invention, the detection
unit may be operably coupled to the information processing system.
Data from the detection unit may be processed by the processor and
data stored in memory. Data on emission profiles for standard
nucleotides may also be stored in memory. The processor may compare
the emission spectra from nucleotides in the flow path and/or
flow-through cell to identify the type of nucleotide released from
the nucleic acid molecule. The memory may also store the sequence
of nucleotides released from the nucleic acid molecule. The
processor may analyze the data from the detection unit to determine
the sequence of the nucleic acid. The information processing system
may also perform standard procedures such as subtraction of
background signals and "base-calling" determination when
overlapping signals are detected.
[0083] 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.
[0084] 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
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 as well as for analysis and reporting of the data
gathered.
[0085] In certain embodiments of the invention, custom designed
software packages may be used to analyze the data obtained from the
detection unit. 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 PRISM.TM.
DNA Sequencing Analysis Software (Applied Biosystems, Foster City,
Calif.), the Sequencher.TM. 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.php.
EXAMPLES
Example 1
Raman Detection of Nucleotides
[0086] Methods and Apparatus
[0087] In a non-limiting example, the excitation beam of a Raman
detection unit was generated by a titanium:sapphire laser (Mira by
Coherent) at a near-infrared wavelength (750.about.950 nm) or a
gallium aluminum arsenide diode laser (PI-ECL series by Process
Instruments) at 785 nm or 830 nm. Pulsed laser beams or continuous
beams were used. The excitation beam was reflected by a dichroic
mirror (holographic notch filter by Kaiser Optical or a dichromatic
interference filter by Chroma or Omega Optical) into a collinear
geometry with the collected beam. The reflected beam passed through
a microscope objective (Nikon LU series), and was focused onto the
Raman active substrate where target analytes (nucleotides or purine
or pyrimidine bases) were located.
[0088] The Raman scattered light from the analytes was collected by
the same microscope objective, and passed the dichroic mirror to
the Raman detector. The Raman detector comprised a focusing lens, a
spectrograph, and an array detector. The focusing lens focused the
Raman scattered light through the entrance slit of the
spectrograph. The spectrograph (Acton Research) comprised a grating
that dispersed the light by its wavelength. The dispersed light was
imaged onto an array detector (back-illuminated deep-depletion CCD
camera by RoperScientific). The array detector was connected to a
controller circuit, which was connected to a computer for data
transfer and control of the detector function.
[0089] For surface-enhanced Raman spectroscopy (SERS), the Raman
active substrate consisted of metallic nanoparticles or
metal-coated nanostructures. Silver nanoparticles, ranging in size
from 5 to 200 nm, was made by the method of Lee and Meisel (J.
Phys. Chem., 86:3391, 1982). Alternatively, samples were placed on
an aluminum substrate under the microscope objective. The Figures
discussed below were collected in a stationary sample on the
aluminum substrate. The number of molecules detected was determined
by the optical collection volume of the illuminated sample.
[0090] The ability to detect single nucleotides by SERS was
confirmed using a 100 .mu.m or 200 .mu.m microfluidic channel. In
various embodiments of the invention, nucleotides may be delivered
to a Raman active substrate through a microfluidic channel (between
about 5 and 200 .mu.m wide). Microfluidic channels can be made by
molding polydimethylsiloxane (PDMS), using the technique disclosed
in Anderson et al. ("Fabrication of topologically complex
three-dimensional microfluidic systems in PDMS by rapid
prototyping," Anal. Chem. 72:3158-3164, 2000).
[0091] Where SERS was performed in the presence of silver
nanoparticles, the nucleotide, purine or pyrimidine analyte was
mixed with LiCl (90 .mu.M final concentration) and nanoparticles
(0.25 M final concentration silver atoms). SERS data were collected
using room temperature analyte solutions.
Results
[0092] Nucleoside monophosphates, purine bases and pyrimidine bases
were analyzed by SERS, using the system disclosed above. Table 1
shows the present detection limits for various analytes of
interest.
TABLE-US-00001 TABLE 1 SERS Detection of Nucleoside Monophosphates,
Purines and Pyrimidines Number of Molecules Analyte Final
Concentration Detected dAMP 9 picomolar (pM) ~1 molecule Adenine 9
pM ~1 molecule dGMP 90 .mu.M 6 .times. 10.sup.6 Guanine 909 pM 60
dCMP 909 .mu.M 6 .times. 10.sup.7 Cyotosine 90 nM 6 .times.
10.sup.3 dTMP 9 .mu.M 6 .times. 10.sup.5 Thymine 90 nM 6 .times.
10.sup.3
[0093] Conditions were optimized for adenine nucleotides only. LiCL
(90 .mu.M final concentration) was determined to provide optimal
SERS detection of adenine nucleotides. Detection of other
nucleotides may be facilitated by use of other alkali-metal halide
salts, such as NaCl, KCl, RbCl or CsCl. The claimed methods are not
limited by the electrolyte solution used, and it is contemplated
that other types of electrolyte solutions, such as MgCl, CaCl, NaF,
KBr, LiI, etc. may be of use. The skilled artisan will realize that
electrolyte solutions that do not exhibit strong Raman signals will
provide minimal interference with SERS detection of nucleotides.
The results demonstrate that the Raman detection system and methods
disclosed above were capable of detecting and identifying single
molecules of nucleotides and purine bases. This is the first report
of Raman detection of unlabeled nucleotides at the single
nucleotide level.
Example 2
Raman Emission Spectra of Nucleotides, Purines and Pyrimidines
[0094] The Raman emission spectra of various analytes of interest
was obtained using the protocol of Example 1, with the indicated
modifications. FIG. 4 shows the Raman emission spectra of a 100 mM
solution of each of the four nucleoside monophosphates, in the
absence of surface enhancement and without Raman labels. No LiCl
was added to the solution. A 100 millisecond (msec) data collection
time was used. Lower concentrations of nucleotides may be detected
with longer collection times. Excitation occurred at 514 nm. For
each of the following figures, a 785 nm excitation wavelength was
used. As shown in FIG. 4, the unenhanced Raman spectra showed
characteristic emission peaks for each of the four unlabeled
nucleoside monophosphates.
[0095] FIG. 5 shows the SERS spectrum of a 1 nm solution of
guanine, in the presence of LiCl and silver nanoparticles. Guanine
was obtained from dGMP by acid treatment, as discussed in Nucleic
Acid Chemistry, Part 1, L. B. Townsend and R. S. Tipson (eds.),
Wiley-Interscience, New York, 1978. The SERS spectrum was obtained
using a 100 msec data collection time.
[0096] FIG. 6 shows the SERS spectrum of a 10 nM cytosine solution,
obtained from dCMP by acid hydrolysis. Data were collected using a
1 second collection time.
[0097] FIG. 7 shows the SERS spectrum of a 100 nM thymine solution,
obtained by acid hydrolysis of dTMP. Data were collected using a
100 msec collection time.
[0098] FIG. 8 shows the SERS spectrum of a 100 pM adenine solution,
obtained by acid hydrolysis of dAMP. Data were collected for 1
second.
[0099] FIG. 9 shows the SERS spectrum of a 500 nM solution of dATP
(lower trace) and fluorescein-labeled dATP (upper trace).
dATP-fluorescein was purchased from Roche Applied Science
(Indianapolis, Ind.). The Figure shows a strong increase in SERS
signal due to labeling with fluorescein.
Example 3
SERS Detection of Nucleotides and Amplification Products
[0100] Silver Nanoparticle Formation
[0101] Silver nanoparticles used for SERS detection were produced
according to Lee and Meisel (1982). Eighteen milligrams of
AgNO.sub.3 were dissolved in 100 mL (milliliters) of distilled
water and heated to boiling. Ten mL of a 1% sodium citrate solution
was added drop-wise to the AgNO.sub.3 solution over a 10 min
period. The solution was kept boiling for another hour. The
resulting silver colloid solution was cooled and stored.
[0102] SERS Detection of Adenine
[0103] The Raman detection system was as disclosed in Example 1.
One mL of silver colloid solution was diluted with 2 mL of
distilled water. The diluted silver colloid solution (160 .mu.L)
(microliters) was mixed with 20 .mu.L of a 10 nM (nanomolar)
adenine solution and 40 .mu.L of LiCl (0.5 molar) on an aluminum
tray. The LiCl acted as a Raman enhancing agent for adenine. The
final concentration of adenine in the sample was 0.9 nM, in a
detection volume of about 100 to 150 femtoliters, containing an
estimated 60 molecules of adenine. The Raman emission spectrum was
collected using an excitation source at 785 nm excitation, with a
100 millisecond collection time. As shown in FIG. 10, this
procedure demonstrated the detection of 60 molecules of adenine,
with strong emission peaks detected at about 833 nm and 877 nm. As
discussed in Example 1, single molecule detection of adenine has
been shown using the disclosed methods and apparatus.
[0104] Rolling Circle Amplification
[0105] One picomole (pmol) of a rolling circle amplification (RCA)
primer was added to 0.1 pmol of circular, single-stranded M13 DNA
template. The mixture was incubated with 1.times. T7 polymerase 160
buffer (20 mM (millimolar) Tris-HCl, pH 7.5, 10 mM MgCl.sub.2, 1 mM
dithiothreitol), 0.5 mM dNTPs and 2.5 units of T7 DNA polymerase
for 2 hours at 37.degree. C., resulting in formation of an RCA
product. A negative control was prepared by mixing and incubating
the same reagents without the DNA polymerase.
[0106] SERS Detection of RCA Product
[0107] One .mu.L of the RCA product and 1 .mu.L of the negative
control sample were separately spotted on an aluminum tray and
air-dried. Each spot was rinsed with 5 .mu.L of 1.times.PBS
(phosphate buffered saline). The rinse was repeated three times and
the aluminum tray was air-dried after the final rinse.
[0108] One milliliter of silver colloid solution prepared as above
was diluted with 2 mL of distilled water. Eight microliters of the
diluted silver colloid solution was mixed with 2 .mu.L of 0.5 M
LiCl and added to the RCA product spot on the aluminum tray. The
same solution was added to the negative control spot. The Raman
signals were collected as disclosed above. As demonstrated in FIG.
11, an RCA product was detectable by SERS, with emission peaks at
about 833 and 877 nm. Under the conditions of this protocol, with
an LiCl enhancer, the signal strength from the adenine moieties is
stronger than those for guanine, cytosine and thymine. The negative
control (not shown) showed that the Raman signal was specific for
the RCA product, as no signal was observed in the absence of
amplification.
Example 4
Exonuclease Digestion of Nucleic Acids
[0109] Exonuclease treatment is performed according to Sauer et al.
(J. Biotech. 86:181-201, 2001). Single nucleic acid molecules
labeled on the 5' end with biotin are prepared by PCR amplification
of a nucleic acid template, using a 5'-biotinylated oligonucleotide
primer. A cone-shaped 3 .mu.m single-mode optical fiber
(SMC-A0630B, Laser Components GmbH, Olching, Germany) is prepared.
The glass fiber is chemically etched with HF to form a sharp tip.
After coating with 3-mercaptopropyltrimethoxysilane, the tip is
treated with .gamma.-maleinimidobutyric acid N-hydroxysuccinamide
(GMBS). The tip of the fiber is activated with streptavidin and
allowed to bind to the biotinylated DNA. Unbound DNA is removed by
washing.
[0110] The fiber containing a single molecule of bound DNA is
inserted into a PDMS reaction chamber attached to a 5 .mu.m
microchannel. Exonuclease I is added to the reaction chamber to
initiate cleavage of the ssDNA. The exonuclease is confined to the
reaction chamber by use of an optical trap (e.g. Walker et al.,
FEBS Lett. 459:39-42, 1999; Bennink et al., Cytometry 36:200-208,
1999; Mehta et al., Science 283:1689-95, 1999; Smith et al., Am. J.
Phys. 67:26-35, 1999). Optical trapping devices are available from
Cell Robotics, Inc. (Albuquerque, N. Mex.), S+L GmbH (Heidelberg,
Germany) and P.A.L.M. Gmbh (Wolfratshausen, Germany). Nucleoside
monophosphates are released by exonuclease digestion and
transported past a Raman detector, as disclosed in Example 1, by
microfluidic flow. A 90 .mu.M concentration of LiCl is added to the
detection mixture, and the microfluidic channel in the vicinity of
the detector is packed with silver nanoparticles prepared according
to Lee and Meisel (1982). Single nucleotides are detected as they
flow past the Raman detector, allowing determination of the nucleic
acid sequence.
Example 5
Nucleic Acid Sequencing Using Raman-Labeled Nucleotides
[0111] Certain embodiments of the invention are exemplified in FIG.
1. FIG. 1 illustrates methods and an apparatus 10 for sequencing
individual single-stranded nucleic acid molecules 13 that are
attached to an immobilization surface 14 in a reaction chamber 11
and disassembled in a deconstruction reaction. In such embodiments
of the invention, the reaction chamber 11 contains one or more
exonucleases 15 that sequentially remove one nucleotide 16 at a
time from the unattached end 17 of the nucleic acid molecule
13.
[0112] As the nucleotides 16 are released, they move down a flow
path 12 past a detection unit 18. The detection unit 18 comprises
an excitation source 19, such as a laser, that emits an excitatory
beam 20. The excitatory beam 20 interacts with the released
nucleotides 16 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, such as a spectrometer, a monochromator
or a charge coupled device (CCD), such as a CCD camera.
Preparation of Reaction Chamber and Flow Path
[0113] 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 (PER-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 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. Methods for making PDMS microchannels are
discussed above.
[0114] 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 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 and the flow path 12 to prevent exonuclease
15 from leaving the reaction chamber 11.
Nucleic Acid Preparation and Exonuclease Treatment
[0115] 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.
[0116] 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 of a sequencing apparatus 10.
[0117] 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.
[0118] Exonuclease 15 activity is initiated by addition of
exonuclease III 15 to the reaction chamber 11. The reaction mixture
is maintained at pH 8.0 and 37.degree. C. As nucleotides 16 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.
Example 6
Nucleic Acid Sequencing Using Covalent Attachment to
Nanoparticles
[0119] Another exemplary embodiment of the invention is disclosed
in FIG. 2. Nucleotides 130 are released from a nucleic acid by
exonuclease activity as discussed above. In certain embodiments of
the invention, the nucleotides 130 are unlabeled. Unlabeled nucleic
acids directly purified from any organ, tissue and/or cell sample
or obtained by known cloning methods may be sequenced using
exonuclease treatment. Released nucleotides 130 travel down a
microfluidic channel 110.
[0120] The released nucleotides 130 are mixed with silver
nanoparticles 140, prepared according to Lee and Meisel (J. Phys.
Chem. 86:3391-3395, 1982). The nanoparticles are 5 to 200 nm in
size. Prior to exposure to nucleotides 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.
The silanized nanoparticles 140 are mixed with nucleotides 130 and
allowed to form covalent cross-links with the nucleotides 130. The
nucleotide-nanoparticle complexes 150 pass through a flow through
cell 170 and are identified by SERS, SERRS and/or CARS using a
Raman detection unit 180. Because of the close proximity of the
nucleotides 130 to the nanoparticles 140, the Raman signals are
greatly enhanced, allowing detection of single nucleotides 130
passing through the flow-through cell 170.
Example 7
Apparatus for Nucleic Acid Sequencing
[0121] FIG. 3 shows another exemplary embodiment of the invention.
A DNA sequencing apparatus 210 comprises a reaction chamber 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 260 is also in fluid
communication with the reaction chamber 220. Nucleotides released
from one or more nucleic acids by exonuclease activity exit the
reaction chamber 220 through the microfluidic channel 260. The
nucleotides are mixed with nanoparticles that move through a
nanoparticle channel 270 in fluid communication with the
microfluidic channel 260. Covalent attachment of nucleotides to
nanoparticles occurs within an attachment channel 280. The
covalently bound nucleotide-nanoparticle complexes pass through a
flow-through cell 290 where the nucleotides are identified by a
Raman detection unit 300. The detection unit 300 comprises a laser
320 and Raman detector 310. The laser emits an excitation beam 330
that excites nucleotides within the flow-through cell 290. Excited
nucleotides emit a Raman signal that is detected by the Raman
detector 310.
[0122] In certain embodiments of the invention, nanoparticles 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, linker compounds and any other
attached or adsorbed molecules. The nanoparticles may be recycled
to a nanoparticle reservoir 370 via a recycling channel 360. In
some embodiments of the invention, nanoparticles 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.
[0123] 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