U.S. patent application number 11/394147 was filed with the patent office on 2007-10-11 for nanowire-based system for analysis of nucleic acids.
Invention is credited to Steven Fung, Hongye Sun, Sam Lee Woo.
Application Number | 20070238186 11/394147 |
Document ID | / |
Family ID | 36829681 |
Filed Date | 2007-10-11 |
United States Patent
Application |
20070238186 |
Kind Code |
A1 |
Sun; Hongye ; et
al. |
October 11, 2007 |
Nanowire-based system for analysis of nucleic acids
Abstract
System for detection and/or analysis of nucleic acids using
nanowires to detect covalent modification of nucleic acids.
Inventors: |
Sun; Hongye; (San Mateo,
CA) ; Fung; Steven; (Palo Alto, CA) ; Woo; Sam
Lee; (Redwood City, CA) |
Correspondence
Address: |
KOLISCH HARTWELL, P.C.
200 PACIFIC BUILDING
520 SW YAMHILL STREET
PORTLAND
OR
97204
US
|
Family ID: |
36829681 |
Appl. No.: |
11/394147 |
Filed: |
March 29, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60666396 |
Mar 29, 2005 |
|
|
|
Current U.S.
Class: |
436/94 ; 340/657;
977/762 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6811 20130101; Y10T 436/14 20150115; C12Q 1/6874 20130101;
Y10T 436/142222 20150115; G01N 27/3276 20130101; G01N 27/3278
20130101; Y10T 436/143333 20150115; C12Q 1/6874 20130101; C12Q
2565/607 20130101; C12Q 2533/101 20130101; C12Q 1/6811 20130101;
C12Q 2565/133 20130101; C12Q 2563/155 20130101 |
Class at
Publication: |
436/094 ;
340/657; 977/762 |
International
Class: |
G01N 33/00 20060101
G01N033/00; G08B 7/06 20060101 G08B007/06 |
Claims
1. A method of detecting and/or analyzing a nucleic acid,
comprising: contacting a nanowire assembly including a nanowire
coupled to a nucleic acid analyte with at least one reagent for
covalent modification of nucleic acids based on nucleic acid
structure; and measuring an electrical characteristic of the
nanowire assembly to determine whether or not the at least one
reagent produced a covalent modification of the nanowire assembly,
thereby providing structural information about the nucleic acid
analyte.
2. The method of claim 1, wherein the step of contacting is
performed on an array of such nanowire assemblies disposed in fluid
communication.
3. The method of claim 2, wherein the nanowire assemblies are
coupled to different nucleic acid analytes, and wherein the step of
measuring provides structural information about each of the
different nucleic acid analytes.
4. The method of claim 1, wherein the step of measuring includes a
step of measuring substantially no change in the electrical
characteristic, thereby indicating that the nucleic acid analyte
lacks structure capable of directing covalent modification by the
at least one reagent.
5. The method of claim 1, wherein the step contacting is performed
on a nanowire assembly including a nucleic acid probe that is
attached to the nanowire and that pairs selectively with the
nucleic acid analyte to couple the nucleic acid analyte to the
nanowire.
6. The method of claim 5, wherein the nucleic acid probe is
configured as a primer of nucleic acid synthesis with the nucleic
acid analyte as a template, and wherein the step of contacting
includes a step of contacting the primer and the template with a
polymerase and a nucleotide substrate for the polymerase.
7. The method of claim 6, wherein the step of measuring determines
an amount of change in the electrical characteristic, if any, the
method further comprising a step of correlating the amount to a
number of nucleotide subunits linked to the primer by the step of
contacting.
8. The method of claim 6, wherein the steps of contacting and
measuring are repeated individually for each of four different
nucleotide substrates corresponding to the nucleotides guanosine,
adenosine, thymidine, and cytidine.
9. The method of claim 8, wherein the steps of contacting and
measuring are repeated over two or more cycles of contacting
individually with the four different nucleotide substrates.
10. The method of claim 1, wherein the step of contacting includes
a step of contacting with a reagent having ligation activity, and
wherein the step of measuring includes a step of determining
whether or not nucleic acid ligation occurred.
11. The method of claim 1, wherein the step of contacting includes
a step of contacting with a reagent for cleavage of nucleic acids,
and wherein the step of measuring includes a step of determining
whether or not nucleic acid cleavage occurred.
12. The method of claim 1, further comprising a step of adjusting a
stringency for nucleic acid hybridization.
13. The method of claim 12, wherein the step of adjusting a
stringency is performed by adjusting at least one of a temperature,
an electric field, and a fluid composition associated with the
nanowire assembly.
14. The method of claim 1, wherein the step of contacting is
performed with a nanowire coupled to a nucleic acid at two or more
spaced positions along the nucleic acid.
15. The method of claim 1, wherein the nanowire functions as a
field effect transistor.
16. A system for detecting and/or analyzing nucleic acids,
comprising: a nanowire assembly including a nanowire coupled to a
nucleic acid template base-paired with a primer; a reagent delivery
system capable of contacting the nanowire assembly sequentially
with different reagents for extension of the primer based on the
nucleic acid template; an electrical detector configured to measure
an electrical characteristic of the nanowire assembly; and a
controller in communication with the reagent delivery system and
the electrical detector and configured to collect data
corresponding to changes in the electrical characteristic in
response to contact with the different reagents, thereby providing
sequence information about the nucleic acid template
17. The system of claim 16, wherein the nanowire assembly is a
plurality of nanowire assemblies coupled to different templates,
and wherein the controller is configured to collect data for each
of the plurality of nanowire assemblies, thereby providing sequence
information about each of the different templates.
18. The system of claim 16, wherein the nanowire assembly functions
as a field effect transistor.
19. The system of claim 16, wherein the different reagents provide
selective addition of guanosine, adenosine, thymidine, and cytidine
to the primer.
20. The system of claim 16, further comprising a heater configured
to apply heat to the nanowire assembly.
Description
CROSS-REFERENCE TO PRIORITY APPLICATION
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Patent Application Ser. No. 60/666,396,
filed Mar. 29, 2006, which is incorporated herein by reference in
it entirety for all purposes.
INTRODUCTION
[0002] Characterization of nucleic acid sequences has widespread
application in a growing number of areas, including clinical
diagnostics and therapeutics, forensics, and analysis of
bio-terrorism agents, among others. For example, the relatively new
field of pharmacogenetics is based on the recognition of a strong
genetic component to the effectiveness of medical treatments. In
particular, nucleic acid sequence differences between members of
the human population can provide much of the variation in response
of the population to a medical treatment, such as a drug. Nucleic
acid sequence analysis of prospective drug recipients thus can be
used to pair each recipient more intelligently with a drug based on
the recipient's genetic makeup. However, current sequencing
technologies may be limited in their ability to meet the growing
demand for sequence information driven by pharmacogenetics and
numerous other applications.
SUMMARY
[0003] The present teachings provide a system for detection and/or
analysis of nucleic acids using nanowires to detect covalent
modification of nucleic acids.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1 is a schematic view of an exemplary system for
nanowire-based detection and/or analysis of nucleic acids, in
accordance with aspects of the present teachings.
[0005] FIG. 2 is a flowchart of an exemplary method of
nanowire-based analysis of nucleic acids, in accordance with
aspects of the present teachings.
[0006] FIG. 3 is a somewhat schematic view of an exemplary array of
nanowire assemblies that can be included in the systems of the
present teachings.
[0007] FIG. 4 is a somewhat schematic view of an exemplary approach
for coupling templates to nanowires to create an array of nanowire
assemblies, in accordance with aspects of the present
teachings.
[0008] FIG. 5 is a somewhat schematic view of an exemplary approach
for coupling primers to nanowires to create an array of nanowire
assemblies, in accordance with aspects of the present
teachings.
[0009] FIG. 6 is a somewhat schematic view of an exemplary system
for nanowire-based sequencing of nucleic acids, in accordance with
aspects of the present teachings.
[0010] FIG. 7 is a flowchart of an exemplary method of
nanowire-based sequencing of nucleic acids, in accordance with
aspects of the present teachings.
[0011] FIG. 8 is a somewhat schematic view of an exemplary approach
to sequencing nucleic acids using an array of nanowire assemblies
and of a graph of exemplary data that may be obtained with this
approach, in accordance with aspects of the present teachings.
[0012] FIG. 9 is a somewhat schematic flowchart of an exemplary
method of nanowire-based analysis of nucleic acids using ligation,
in accordance with aspects of the present teachings.
[0013] FIG. 10 is a somewhat schematic flowchart of an exemplary
method of nanowire-based analysis of nucleic acids using cleavage,
in accordance with aspects of the present teachings.
[0014] FIG. 11 is a somewhat schematic view of an exemplary
nanowire assembly during coupling of a nucleic acid (an analyte
and/or a probe) to a nanowire of the assembly at two or more spaced
sites along the nucleic acid, in accordance with aspects of the
present teachings.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0015] The present teachings provide a system for detection and/or
analysis of nucleic acids using nanowires to detect covalent
modification of nucleic acids. Each nanowire can be coupled to a
nucleic acid analyte and a nucleic acid probe base-paired with the
analyte, to form a nanowire assembly. The nanowire assembly or any
array of nanowire assemblies can be contacted with a reagent for
covalent modification of nucleic acids based on nucleic acid
structure. For example, the reagent can include a nucleic-acid
modifying enzyme, such as a polymerase, a ligase, or a nuclease,
among others, capable of lengthening or shortening nucleic acids. A
change in the size of a nucleic acid(s) coupled to a nanowire
assembly can change the electrical characteristics, such as the
conductance, of the nanowire assembly. Accordingly, a detector can
be used to measure an electrical characteristic of the nanowire
assembly, to determine whether or not the electrical characteristic
has been changed by action of the reagent. The presence or absence
of change in the electrical characteristic (and/or the size (and/or
polarity) of any change) thus can provide structural information,
such as sequence information, about the analyte.
[0016] Overall, nanowire-based analysis of nucleic acids can have a
number of advantages over other systems. These advantages can
include increased sensitivity, analysis of smaller amounts of
analytes, multiplexed analysis of analytes, performance of a large
number of analyses (e.g., hundreds or thousands) in a small space,
decreased size of instrumentation, improved portability, and/or the
like. For example, in contrast to chain termination approaches to
sequencing (e.g., dideoxynucleotide-based sequencing), the
nanowire-based systems described herein can sequence nucleic acids
by successive primer extension and measurement. Accordingly, the
same individual primer and template molecules can be involved in
determining the identity of different nucleotides in the template,
thereby substantially reducing the number of primer and template
molecules necessary for sequencing.
[0017] FIG. 1 shows an exemplary system 20 for nanowire-based
analysis of nucleic acids. The system can include a reagent
delivery system 22, a nanowire assembly 24, a detector 26, and/or a
controller 28.
[0018] Reagent delivery system 22 can transfer one or more reagents
30 for nucleic acid modification, and particularly fluid reagents,
to and/or from the nanowire assembly, shown at 23. The reagent
delivery system can be a flow-based system including a pump(s), a
valve(s), one or more reservoirs, a channel(s) in which the
nanowire assembly is disposed, and/or the like. Further aspects of
the reagent delivery system are described, for example, in Section
V and in Example 3.
[0019] Nanowire assembly 24 can provide a site for nucleic acid
modification. In particular, the nanowire assembly can include a
nanowire 32 and one or more nucleic acids 34 coupled to the
nanowire, shown at 36. The nucleic acids can include an analyte 38,
which is the subject of the analysis, and a probe 40, which
facilitates structural analysis of the analyte. The probe can be
configured to form base pairs with the analyte, shown at 42, so
that the probe can couple the analyte to the nanowire, as in the
present illustration, or vice versa, among others. In some
embodiments, the system can include a plurality of nanowire
assemblies, to form an array of nanowires (and nanowire
assemblies). Further aspects of nanowires, nanowire arrays, nucleic
acids, and coupling nucleic acids to nanowires are described, for
example, in Sections I and II and in Examples 1 and 2, among
others.
[0020] Detector 26 can measure a characteristic such as an
electrical characteristic of the nanowire assembly. Accordingly,
the detector can be coupled electrically to the nanowire, shown at
44, for example, through a pair of electrodes disposed at spaced
positions along the nanowire. In some examples, the detector can
measure electrical characteristics from each of a plurality of
nanowires disposed in an array, for multiplexed analysis of nucleic
acids. Further aspects of detectors are described, for example, in
Section IV.
[0021] Controller 28 can control various aspects of system
operation. For example, the controller can be coupled to the
detector, shown at 46, to determine when the detector measures the
electrical characteristic and/or on which nanowire assembly of an
array. The controller also can store and/or process data received
from the detector, such as data corresponding to measured
electrical characteristics. The controller also, or alternatively,
can be coupled to the reagent delivery system, shown at 48, to
control and/or monitor delivery of reagents to/from the nanowire
assembly. Further aspects of controllers are described, for
example, in Section VI.
[0022] FIG. 2 is a flowchart 60 illustrating an exemplary method of
nanowire-based analysis of nucleic acids. The method can include
steps of (1) providing a nanowire assembly, shown at 62, (2)
contacting with nanowire assembly with at least one reagent, shown
at 64, and (3) measuring an electrical characteristic of the
nanowire assembly, shown at 66. These steps can be performed in any
suitable order, in any suitable combination, and any suitable
number of times.
[0023] A nanowire assembly can be provided. The nanowire assembly
can include a nucleic acid analyte coupled to a nanowire, either
directly and/or through a nucleic acid probe that pairs
(hybridizes) with the analyte. The step of providing can include or
be preceded by a step of forming a nanowire assembly. The step of
forming a nanowire assembly can include coupling nucleic acids
covalently and/or noncovalently to a nanowire. In some examples, an
array of nanowire assemblies can be provided. Further aspects of
providing and forming a nanowire assembly are described, for
example, in Sections I-III and in Examples 1 and 2.
[0024] The nanowire assembly can be contacted with a reagent for
covalent modification of nucleic acids based on nucleic acid
structure. For example, the reagent can include an enzyme (such as
a polymerase, ligase, nuclease, etc.) and/or a nucleotide monomer
or polymer, among others. A covalent modification can include
nucleotide addition to, or removal from, the analyte and/or probe.
Accordingly, the structure of the analyte can determine, for
example, presence or absence of the covalent modification, the
position of the covalent modification (such as position within the
analyte and/or probe), and/or the extent of the covalent
modification (such as number of nucleotides added or removed).
Further aspects of reagents and covalent modification of nucleic
acids are described, for example, in Section V and in Examples 3-5,
among others.
[0025] An electrical characteristic of the nanowire assembly can be
measured. The electrical characteristic, and particularly a change
in the electrical characteristic, if any, can be used to determine
whether or not the analyte has a structure that allows covalent
modification by the reagent, thus providing information about the
analyte's structure. Further aspects of measuring electrical
characteristics and determining analyte structure based on changes
in an electrical characteristic are described, for example, in
Section IV and in Examples, 3-5, among others.
[0026] The steps of contacting and measuring can be repeated any
suitable number of times. In some examples, contacting and
measuring can be repeated with different reagents (such as
different enzymes and/or nucleotide substrates) and/or can be
repeated cyclically with the same substrate multiple times, such as
for determining the sequence of an analyte region of two or more
nucleotides.
[0027] The methods of the present teachings also or alternatively
can include a step of adjusting the stringency of base-pair
interactions. The stringency can be adjusted to alter the stability
of nucleic acid strand-strand interactions, such as to increase or
decrease the total number, uninterrupted length, and/or type of
base-pair interactions (e.g., G-C base pairs are generally more
stable than A-T base pairs) necessary to hold together nucleic acid
strands that are complementary. In some examples, adjusting the
stringency can include disrupting base-pair interactions of nucleic
acid duplexes, such that only less stable duplexes and/or at least
substantially all double-stranded duplexes are disrupted to form
unpaired single strands. Adjusting stringency can be performed
electrically (e.g., by positively or negatively biasing a nanowire
assembly), by changing the temperature of the nanowire assembly
(e.g., by heating or cooling a corresponding reaction compartment
and/or fluid disposed in, or destined for, the compartment), and/or
chemically (e.g., by adjusting ionic strength, the concentration of
divalent or multivalent cations and/or anions, the solvent
dielectric constant (e.g., by changing the concentration of
dimethylformamide or another organic solvent), and/or enzymatically
(e.g., by adding or adjusting the concentration of enzymes such as
helicase that favor the pairing or unpairing of nucleic acid bases
or strands), and/or changing the concentration of chaotropic agents
(such as urea), among others). Nucleic acid duplexes can be
disrupted, for example, after contacting and before measuring
(e.g., to remove a contribution to the electrical characteristic
produced by nucleic acid duplexes), or after contacting and after
measuring (e.g., to facilitate performance of another cycle of
contacting and measuring). In some examples, adjusting the
stringency can include reducing the ionic strength of fluid in
contact with the nanowire assembly to increase the sensitivity of
measuring the electrical characteristic.
[0028] Further aspects of the present teachings are described in
the following sections, including (I) nanowires; (II) nucleic
acids, including (A) analytes and (B) probes; (III) nanowire
assemblies; (IV) detectors; (V) reagent delivery systems; (VI)
controllers; and (VII) examples.
I. NANOWIRES
[0029] The systems of the present teachings include one or more
nanowires. A nanowire, as used herein, is an elongate semiconductor
having a sub-micrometer cross-sectional dimension at one or more
(or all) positions along its length. The cross-sectional dimension
(and/or orthogonal cross-sectional dimensions) can be less than
about 500 nm, 100 nm, 20 nm, 5 nm, or 1 nm, among others.
[0030] The nanowires can have any suitable length. Exemplary
lengths include at least about 1 .mu.m, 5 .mu.m, or 20 .mu.m, among
others. Furthermore, the nanowires can have any suitable aspect
ratio (length relative to a cross-sectional dimension (and/or
relative to orthogonal cross-sectional dimensions) to produce an
elongate structure. Exemplary aspect ratios include at least about
2:1, 10:1, 100:1, or 1000:1.
[0031] The nanowires can have any suitable cross-sectional shape.
Exemplary cross-sectional shapes include circular, elliptical,
polygonal (triangular, rectangular, etc.), irregular, and/or a
combination thereof. In some examples the nanowires can be
nanotubes having a hollow core.
[0032] The nanowires can be formed of any suitable material(s). For
example, the nanowires can be formed of semiconductor materials
(elements or alloys), with or without a dopant. Exemplary
semiconductor materials to form the body (or a coating or lining)
of the nanowire include silicon, germanium, and/or carbon, among
others. Exemplary dopants include n-type dopants and/or p-type
dopants, such as nitrogen and phosphorus, respectively, among
others. Other materials that can be suitable to form the body
(and/or coating or lining) of the nanowires or as dopants therein
are described in the following patent applications, which are
incorporated herein by reference: Ser. No. 09/935,776, filed Aug.
22, 2001 (Pub. No. US 2002/0130311 A1); Ser. No. 10/020,004, filed
Dec. 11, 2001 (Pub. No. US 2002/0117659 A1); Ser. No. 10/033,369,
filed Oct. 24, 2001 (Pub. No. US 2002/0130353); and Ser. No.
10/196,337, filed Jul. 16, 2002 (Pub. No. US 2003/0089899 A1).
II. NUCLEIC ACIDS
[0033] The systems of the present teachings provide nanowires
coupled to nucleic acids. A nucleic acid (or an oligonucleotide, an
oligomer, or a polynucleotide), as used herein, is a polymer of at
least two nucleotide subunits linked together. The nucleic acid can
be single-stranded or double-stranded (a duplex), among others.
Double-stranded nucleic acids generally are formed by
hydrogen-bonding (base-pairing) between aligned nucleotides of
paired strands of nucleic acids, for example, adenosine (A) paired
with thymidine (T) (or uridine (U) in RNA), and guanosine (G)
paired with cytidine (C), among others.
[0034] The nucleic acid can have any suitable natural and/or
artificial structure. The nucleic acid can include a
sugar-phosphate backbone of alternating sugar and phosphate
moieties, with a nucleotide base attached to each sugar moiety. Any
sugar(s) can be included in the backbone including ribose (for
RNA), deoxyribose (for DNA), arabinose, hexose, 2'-fluororibose,
and/or a structural analog of a sugar, among others. The nucleotide
base can include, for example, adenine, cytosine, guanine, thymine,
uracil, inosine, 2-amino adenine, 2-thiothymine, 3-methyl adenine,
C5-bromouracil, C5-fluorouracil, C5-iodouracil, C5-methyl cytosine,
7-deazaadeine, 7-deazaguanine, 8-oxoadenine, 8-oxoguanine,
2-thiocytosine, or the like. The nucleic acids of the present
teachings can include any other suitable alternative backbone.
Exemplary alternative backbones include phosphoramides,
phosphorothiozates, phosphorodithioates, O-methylphosphoroamidites,
peptide nucleic acids, positively charged backbones, non-ribose
backbones, etc. Nucleic acids with artificial backbones and/or
moieties can be suitable, for example, to increase or reduce the
total charge, increase or reduce base-pairing stability, increase
or reduce chemical stability, to alter the ability to be acted on
by a reagent, and/or the like.
[0035] A nanowire can be coupled to a nucleic acid analyte (or a
plurality of structurally different analytes) and/or to a nucleic
acid probe (or a plurality of structurally different probes).
Furthermore, the nanowire can be coupled to a single molecule or to
a plurality of molecules of each analyte and/or probe.
A. Analytes
[0036] An analyte, as used herein, is a nucleic acid that is the
subject of a nanowire-based analysis. The analyte can be from any
suitable source, can have any suitable structure, and can be
analyzed for any suitable feature. In some examples, the analyte
can be a template, that is, a nucleic acid used as a model or guide
for forming at least a region of another nucleic acid. The template
can, for example, direct addition of one or more nucleotides to a
probe, serially or in parallel, according to a complementary region
of the template that base-pairs with the one or more
nucleotides.
[0037] The analyte can be from any suitable source. Exemplary
sources can include a human subject, a nonhuman animal, a plant, a
microorganism, a research sample, an environmental sample (such as
soil, air, water, etc.), and/or in vitro synthesis, among
others.
[0038] The human subject can be a disease patient, a genetic
screening subject, a person to be identified, a forensic subject,
and/or the like. The analyte can be obtained from any suitable site
in the human subject, including a sample from blood, plasma, serum,
sperm, urine, sweat, tears, sputum, mucus, milk, a tissue sample, a
tumor biopsy, cultured cells, and/or the like.
[0039] The analyte can be obtained in any suitable form by any
suitable processing. For example, the analyte can be included in a
crude lysate or can be a purified analyte obtained, for example, by
ion exchange chromatography, selective precipitation,
centrifugation, and/or amplification (such as with the polymerase
chain reaction), among others. The analyte can be single- or
double-stranded and can have any suitable size. In some examples,
the analyte can have a single size or a set of sizes produced by
shearing, restriction endonuclease digestion, in vitro synthesis,
amplification, limited chemical digestion, and/or the like. In some
examples, the analyte coupled to a nanowire includes a plurality of
discrete strands of similar or identical length and sequence
content, or of distinct lengths and/or sequence content. Strands of
distinct length can be overlapping fragments, for example,
including a common region of similar or identical sequence, such as
for hybridization (base-pairing) with a probe. The analyte can be
any suitable size relative to the probe. In some examples, the
analyte is about the same size as the probe. In some examples, the
analyte is longer than the probe and can be substantially longer
than the probe, such as at least about two, ten, or one hundred
times as long.
[0040] The analyte can be analyzed to obtain structural information
about any suitable feature(s). Generally, the structural
information relates to a sequence feature. The sequence feature can
be defined by any suitable length of nucleotides. The structural
information thus can be the presence or absence of a sequence
feature of interest, the nucleotide identity (e.g., G, A, T, or C)
at a particular position(s) within the analyte (e.g., to
characterize a single nucleotide corresponding to a single
nucleotide polymorphism in the population), and/or the particular
sequence of a stretch of at least about 5, 10, 50, 200, or 1000
nucleotides, among others, of the analyte. The sequence feature
thus can be compared to a known sequence, to look for nucleotide
identity and/or differences, or can correspond to a previously
unsequenced region of a genome or other polynucleotide
structure.
B. Probes
[0041] A probe, as used herein, is a nucleic acid that facilitates
analysis of the nucleic acid analyte. The probe can be from any
suitable source, can have any suitable structure, and can be used
to analyze the analyte for any suitable feature(s).
[0042] The probe can be obtained from a natural and/or artificial
source. Accordingly, the probe can be synthesized or formed by a
cell(s), a cell lysate(s), a synthetic enzyme(s), chemical
synthesis, enzymatic cleavage, chemical cleavage, and/or ligation,
among others. The probe thus can be RNA, DNA, or any suitable
artificial derivative thereof. Furthermore, the probe can belong to
the same structural class of molecules as the analyte (e.g., each
being DNA or each being RNA) or to a different class of molecules
(e.g., the probe being DNA and the analyte RNA (or vice versa), or
the probe having an uncharged or positively charged backbone and
the analyte having a phosphodiester backbone, among others).
[0043] The probe can have any suitable structure relative to the
analyte. The probe can be configured to form a duplex with the
analyte through base-pair interactions, so that the probe and
analyte together form an at least partially double-stranded nucleic
acid. Accordingly, a section (or all) of the probe can be
complementary to a section (or all) of the analyte. Alternatively,
or in addition, the probe can include a double-stranded region,
independent of the analyte, for example, to couple the probe to a
nanowire. The probe can be configured to hybridize (base-pair) to
any region of the analyte, for example, the probe can hybridize
adjacent an end or spaced from the end of the analyte. In some
examples, the probe can be a primer. The primer can be configured
so that the 3'-end of the probe is base-paired with the analyte and
spaced from the ends of the analyte, allowing the 3'-end to be
extended with a polymerase (or ligase) and a suitable nucleotide
substrate(s). The probe can have any suitable length sufficient to
form a duplex structure with another nucleic acid, particularly the
analyte. The duplex structure can be stabilized, for example, by
nucleotide addition to the probe (such as with a polymerase or
ligase, among others).
[0044] The systems of the present teachings can include one probe
or a plurality of probes. The plurality of probes can be configured
to form duplexes with different analytes, different regions of the
same analyte, or with the same region of the same analyte (e.g.,
see Example 4).
III. NANOWIRE ASSEMBLIES
[0045] The systems of the present teachings can include one or more
nanowire assemblies. The nanowire assemblies can be disposed in any
suitable arrangement, can include any suitable number and type of
nucleic acids, and can couple the nucleic acids to nanowires by any
suitable mechanism(s).
[0046] The systems of the present teachings can include an array of
nanowire assemblies. The nanowire assemblies can be arrayed in a
linear arrangement, a two-dimensional arrangement, and/or a
three-dimensional arrangement (such as stacked two-dimensional
arrays). The array can include any suitable number of assemblies,
including at least about ten, one-hundred, or one-thousand, among
others. The nanowire assemblies can include a different probe (or
probes) or the same probe (or probes) in each assembly, and/or a
different analyte (or analytes) or the same analyte(s) or analyte
region(s) in each assembly. Accordingly, the nanowire assemblies
can be configured to analyze distinct regions (nonoverlapping or
overlapping) of the same analyte, the same region of the same
analyte, and/or different regions of different analytes.
[0047] The analyte and probe can be coupled to each nanowire by any
suitable mechanism. The analyte and/or the probe can be coupled
directly to the nanowire by a covalent or noncovalent mechanism.
Covalent mechanisms include bond formation between any suitable
reactive pair with pair members disposed on the nanowire and a
nucleic acid. An exemplary mechanism includes reaction of
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
with nucleic acids to form nucleic acid derivatives that can react
with activated nanowire surfaces. Alternatively, or in addition,
nucleic acids can be bound to nanowires through noncovalent
specific binding pair interactions. For example, a first member of
a specific binding pair can be attached to a nanowire and a second
member of the specific binding pair can be attached (or included
in) a nucleic acid analyte and/or probe. Specific binding pairs
generally undergo specific binding, that is, binding to one another
to the exclusion of binding to most other moieties. Specific
binding can be characterized by a dissociation constant or
coefficient (alternatively termed an affinity or binding constant
or coefficient). Generally, dissociation constants for specific
binding range from 10.sup.-4 M to 10.sup.-12 M and lower, and
preferred dissociation constants for specific binding range from
10.sup.-8 or 10.sup.-9 M to 10.sup.-12 M and lower. Exemplary
specific binding pairs are presented in Table 1: TABLE-US-00001
TABLE 1 Exemplary Specific Binding Pairs Specific Binding Member
Partner cell-surface receptor secreted hormone or cell-associated
ligand nuclear receptor nuclear hormone or DNA antibody antigen
avidin or streptavidin biotin lectin or carbohydrate receptor
carbohydrate DNA antisense DNA; protein RNA antisense or other RNA;
protein enzyme enzyme substrate or regulator histidine NTA
(nitrilotriacetic acid) IgG protein A or protein G
Specific binding pair interactions (e.g., base pairing) also can be
used to associate the analyte and probe with each other.
Accordingly, the probe (or analyte) can be coupled more directly to
the nanowire than the analyte (or probe) with which it is base
paired. Moreover, the probe and analyte can be coupled to a
nanowire at the same time (e.g., in a base-paired condition) or
sequentially, for example, by coupling the probe first and then the
analyte, or vice versa (see Example 2). Furthermore, the probe
and/or analyte can be coupled to the nanowire at a single site or
at multiple sites (e.g., spaced sites) along each molecule of the
probe/analyte (see Example 6).
[0048] Probes and/or analytes can contact nanowires to form
nanowire assemblies at any suitable time using any suitable contact
mechanism. In some examples, the nanowires can be disposed in an
array and then different probes and/or analytes selectively
contacted with (and coupled to) individual nanowires or subsets of
nanowires in the array. For example, individual probes and/or
analytes (or different sets thereof) can be selectively dispensed
to regions of the array in small droplets of fluid so that the
regions remain in fluid isolation, such as by inkjet printing
technology (e.g., using a dispensing head with thin-film heater
elements and/or piezoelectric elements, among others, as used in
inkjet printheads). Alternatively, or in addition, the array of
nanowires can be contacted with the probes and/or analytes with the
nanowires disposed in fluid communication. For example, the probes
and/or analytes can selectively interact with the nanowires based
on specific binding partners (and particularly nucleic acids)
previously coupled to the nanowires.
[0049] Further aspects of nanowires, nanowire assemblies, and
assays that may be performed with nanowires are described in the
following patent applications, which are incorporated herein by
reference: U.S. Provisional Patent Application Ser. No. 60/612,315;
U.S. patent application Ser. No. 09/935,776, filed Aug. 22, 2001
(Pub. No. US 2002/0130311 A1); U.S. patent application Ser. No.
10/020,004, filed Dec. 11, 2001 (Pub. No. US 2002/0117659 A1); U.S.
patent application Ser. No. 10/033,369, filed Oct. 24, 2001 (Pub.
No. US 2002/0130353); and U.S. patent application Ser. No.
10/196,337, filed Jul. 16, 2002 (Pub. No. US 2003/0089899 A1).
IV. DETECTORS
[0050] The systems of the present teachings generally include one
or more detectors (also termed sensors) to measure an electrical
characteristic of each nanowire (and nanowire assembly) of a
nanowire array. In some examples, the detector is coupled
electrically to each nanowire in a serial fashion, using, for
example, electronic switching devices. Accordingly, the detector
can be coupled to the nanowires in a repeatable cycle, and the
electrical characteristic of each nanowire can be detected
periodically to measure any time-dependent (and generally
reagent-dependent) changes (if any) in the electrical
characteristic.
[0051] The detector can measure any suitable electrical
characteristic. Exemplary electrical characteristics include
conductance, resistance, current, voltage, and/or the like. The
electrical characteristic can be measured qualitatively (e.g.,
change or no change and/or a positive or negative change) or
quantitatively (e.g., to determine a magnitude of the change, if
any). The electrical characteristic can provide an analog and/or
digital output.
V. REAGENT DELIVERY SYSTEMS
[0052] The systems of the present teachings can include one or more
reagent delivery systems. The reagent delivery systems can include
one or more pumps, valves, fluid reservoirs, channels, and/or
reagents, among others.
[0053] Pumps generally include any mechanism for moving fluid
and/or reagents disposed in fluid. In some examples, the pump can
be configured to move fluid and/or reagents through passages with
small volumes (i.e., microfluidic structures). The pump can operate
mechanically by exerting a positive or negative pressure on fluid
and/or on a structure carrying fluid, electrically by appropriate
application of an electric field(s), or both, among others.
Exemplary mechanical pumps may include syringe pumps, peristaltic
pumps, rotary pumps, pressurized gas, pipettors, etc. The
mechanical pumps may be micromachined, molded, etc. An exemplary
peristaltic pump created with a fluidic layer and a control layer
that are elastomeric is described, for example, in U.S. Pat. No.
6,408,878, issued Jun. 25, 2002, which is incorporated herein by
reference. Exemplary electrical pumps can include electrodes and
may operate by electrophoresis, electroendoosmosis,
electrocapillarity, dielectrophoresis (including traveling wave
forms thereof), and/or the like.
[0054] Valves generally include any mechanism for regulating the
passage of fluid through a channel. The valves can include, for
example, deformable members that can be selectively deformed to
partially or completely close a channel, a movable projection that
can be selectively extended into the channel to partially or
completely block the channel, an electrocapillary structure, and/or
the like. The valves can be operable, for example, to select a
reagent to be contacted with a nanowire assembly (or assemblies),
from a set of available reagents. Accordingly, the valves can be
operable to provide selective fluid communication between a
nanowire assembly (or assemblies) in a channel (a reaction
compartment) and two or more reagent reservoirs.
[0055] Fluid reservoirs generally include any compartments for
holding reagents before and/or after they have passed through the
reaction compartment holding one or more nanowire assemblies. The
fluid reservoirs can have any suitable volume. In some examples,
the fluid reservoirs have a volume that is substantially larger
than the volume of the reaction compartment, such as a volume that
is at least about ten-fold, one-hundred-fold, or one-thousand fold
the reaction compartment volume. The fluid reservoirs can be
configured to be accessible from outside the system, to facilitate
adding or removing fluid, such as with a pipette.
[0056] Channels generally include any passages that allow flow of
fluid and/or movement of reagents. The channels can extend, for
example, between fluid reservoirs of the system, and can define a
reaction compartment that holds the nanowire assembly (or
assemblies). The channels can have any suitable dimensions. In some
embodiments, the channels can be microfluidic channels.
Microfluidic channels or compartments, as used herein, can have a
cross-sectional dimension, at one or more positions along their
length, of less than about one micrometer or less than about 100
nanometers.
[0057] Reagents can have any suitable function in nucleic acid
analysis. The reagents can be configured, for example, to
structurally modify nucleic acids of nanowire assemblies, to wash
out (remove) a previously dispensed reagent and/or a released
nucleic acid, to adjust the stringency of hybridization between
paired nucleic acid strands, to disrupt base-pair interactions
(denature duplexes into single strands) substantially or
completely, and/or the like.
[0058] Reagents include any chemical substances that can contact
nanowire assemblies to facilitate analysis of nucleic acids. The
chemical substances can be present in any suitable form in a
reagent, including as a mixture, a complex, a solution, a
suspension, and/or the like. Exemplary reagents can include
catalysts and/or mono- and/or polynucleotides. Other exemplary
reagent components can include carrier fluids (such as water and/or
an organic fluid), enzyme cofactors (such as divalent cations
(e.g., magnesium, zinc, manganese, etc.), ribonucleoside
triphosphates (such as adenosine triphosphate (ATP)), S-adenosyl
methionine (SAM), etc.), reducing agents (such as dithiothreitol
(DTT), beta-mercaptoethanol, etc.), salts (e.g., to adjust ionic
strength), stabilizing agents (such as serum albumin (e.g., BSA),
size-based exclusion polymers (such as polyethylene glycol (PEG)),
and/or the like.
[0059] Catalysts can include any material that can increase the
rate of a chemical reaction (particularly a reaction that modifies
nucleic acids) without being consumed or produced by the reaction.
Exemplary catalysts for nucleic acid modification are proteins
(enzymes). Any suitable enzymes can be included in reagents.
Exemplary enzymes can add single nucleotides or polynucleotides
covalently to a nucleic acid, generally based on the sequence of a
partner strand based-paired with the nucleic acid (i.e., templated
addition). Single-nucleotide addition enzymes, which add individual
nucleotides successively, generally include polymerases, such as
DNA polymerases (e.g., DNA Polymerase I (or fragments/derivatives
thereof (such as the Klenow fragment)), thermostable DNA
polymerases (such as Taq Polymerase, Vent Polymerase, Pfu
Polymerase, etc.), and/or the like), RNA polymerases (such as
phage-derived polymerases (such as SP6, T7, or T3 RNA polymerase),
reverse transcriptases that add deoxyribonucleotides based on an
RNA template, and/or the like. Polynucleotide addition enzymes,
which add two or more nucleotides at the same time to a nucleic
acid, can include ligases (such as T4 DNA ligase, Taq DNA Ligase,
E. coli DNA Ligase, etc.). Other exemplary enzymes can include
nucleases (cleavage enzymes), such as restriction enzymes,
ribonucleases or deoxyribonucleases (e.g., single- or double-strand
specific enzymes), and/or the like. Other exemplary catalysts can
include polynucleotides (e.g., RNA), synthetic polymers, transition
metal complexes, reactive surfaces, etc.
[0060] The reagents can include one or more mono- or
polynucleotides for contact with nanowire assemblies.
Mononucleotide reagents can include, for example, nucleoside
triphosphates, including deoxyribonucleoside triphosphates (dNTPs)
(e.g., dATP, dCTP, dGTP, dTTP, etc.), ribonucleoside triphosphates
(NTPS) (e.g., ATP, CTP, GTP, UTP, etc.), and/or mixtures thereof,
among others. Polynucleotide reagents can include, for example,
nucleic acid dimers, trimers, tetramers, etc. The polynucleotide
reagents can be configured to be partially or completely
complementary to a region of the analyte (or to a region being
tested for its presence or absence in the analyte). In some
examples, the polynucleotide reagents can include a 5'-phosphate
for ligation to a 3'-hydroxyl of a probe.
VI. CONTROLLERS
[0061] The systems of the present teachings can include at least
one controller. The controller can interface with (control,
coordinate, and/or record) various portions of the systems. For
example, the controller can interface with operation of the reagent
delivery system and/or the detector, among others.
[0062] The controller can interface with any suitable aspect of the
reagent delivery system. In some examples, the controller can
control operation of the pump(s), such as determining when the pump
is operated, the rate of pump operation, selection of a subset of
pumps that are operated, etc. Alternatively, or in addition, the
controller can control operation of valves, to determine, for
example, which reagent(s) are selected for addition to a reaction
compartment, in what order, at what rate, and/or for how long,
among others. The controller also can receive inputs from a user
for user preferences related to operation of the pumps and/or
valves, and/or can store and/or report data related to aspects of
reagent delivery.
[0063] The controller can interface with any suitable aspects of
the detector(s). In some examples, the controller can control
operation of the detector(s), such as determining when the detector
is operated and/or which nanowire assembly is electrically coupled
to the detector at a given time and/or in what order, the size and
timing of a back gate voltage applied to a substrate supporting a
nanowire assembly (or assemblies), a voltage and/or current applied
to the nanowire assembly, and/or the like. The controller also can
receive inputs from a user for user preferences related to
operation of the detector(s), and/or can collect, store, and/or
report data related to aspects of detection.
VII. EXAMPLES
[0064] The following examples describe selected aspects and
embodiments of systems for nanowire-based analysis of nucleic
acids. These examples are included for illustration and are not
intended to limit or define the entire scope of the present
teachings.
Example 1
Nanowire Arrays
[0065] This example describes an exemplary array of nanowire
assemblies; see FIG. 3.
[0066] Nanowire array 80 can include a plurality of nanowire
assemblies 82 supported by a substrate 84. The nanowire assemblies
can be disposed in a linear array on the substrate, as shown here,
and/or in a two- (or three-) dimensional array.
[0067] Each nanowire assembly can include a nanowire 86 extending
between spaced electrodes 88. The nanowire can be electrically
coupled to the electrodes, so that current can pass between the
electrodes through the nanowire. Exemplary electrodes can be formed
of an electrically conductive material, generally a conductive
metal(s) or metal alloy. In exemplary embodiments, the electrodes
can be formed of gold. Electrodes can be formed on the substrate
before or after placement of the nanowires. In exemplary
embodiments, the electrodes can be formed by photolithography
and/or ion beam lithography.
[0068] The substrate can have any suitable composition and
structure. In some embodiments, the substrate can be generally
planar, and can include an electrical insulator layer (such as
silicon dioxide) disposed over a semiconductor layer (such as
silicon). The semiconductor layer can be used to apply a back gate
voltage when the nanowire assembly functions as a field effect
transistor (FET), with the electrodes acting as source and
drain.
[0069] Each nanowire assembly can include nucleic acids 92 coupled
to a nanowire. The nucleic acids can include a probe 94 (such as a
primer) and an analyte 96 (such as a template) base-paired with the
probe. The assemblies (or subsets of two or more of the assemblies)
can include the same probe or can include different probes, as
shown in the present illustration. The assemblies (or subsets of
two or more of the assemblies) can include the same analyte (or
analyte region), as shown in the present illustration, or different
analytes.
Example 2
Coupling Nucleic Acids to Nanowires
[0070] This example describes exemplary approaches for coupling
nucleic acids to nanowires; see FIGS. 4 and 5.
[0071] FIG. 4 shows an exemplary array 100 of nanowire assemblies
102 prepared to receive template 104, shown by the arrow at 106.
Each nanowire assembly can include a nanowire 108 and a different
probe 110 coupled to the nanowire. The probes can be coupled to the
nanowires before or after they are disposed in the array. Templates
104 can be placed in contact with the array and allowed to
hybridize with the probes and/or can be coupled directly to the
nanowires. Accordingly, the probes can be configured to select
complementary templates from a nucleic acid mixture of templates
and nontemplate species. After hybridization, unpaired templates
(and nontemplate species) can be removed.
[0072] FIG. 5 shows an exemplary array 120 of nanowire assemblies
122 prepared to receive primers 124, shown by the arrow at 126.
Each nanowire assembly can include a nanowire 128 and a different
(or the same) template 130 coupled to the nanowire. The templates
can be coupled to the nanowires before or after the nanowires are
disposed in the array. Primers 124 can be placed in contact with
the array and allowed to hybridize with the templates and/or can
couple directly to the nanowires. Accordingly, the templates can be
configured to select complementary primers from a primer mixture.
After hybridization, unpaired (e.g., excess) primer molecules can
be removed.
Example 3
System for Nanowire-Based Sequencing
[0073] This example describes an exemplary system, including
apparatus, method, and data, for nanowire-based sequencing; see
FIGS. 6-8.
[0074] A system 140 for nanowire based sequencing can include a
reagent delivery system 142 in fluid communication with a reaction
compartment 144 holding an array 146 of nanowire assemblies 148.
The system also can include a detector 150 electrically coupled (or
couplable) to the array of nanowire assemblies, and a controller
152 in communication with the detector and the reagent delivery
system.
[0075] Reagent delivery system 142 is configured to move fluid
through reaction compartment 144 for contact with array 146. The
reagent delivery system thus can include a plurality of fluid
reagents 154 for covalent modification of nucleic acids, such as
four different nucleoside triphosphates (dNTPs) 156, 158, 160, and
162, each disposed in an aqueous solution with DNA polymerase
molecules 164. The fluid reagents also can include a wash reagent
166, which can be used, for example, to wash dNTP reagents out of
the reaction compartment after they have contacted the nanowire
assemblies. Movement of the fluid reagents can be driven by a pump
168 disposed, for example, downstream of the fluid reagents. The
pump can drive each fluid reagent to (and past) the reaction
compartment. Fluid reagents can be selected according to selective
opening and closing of valves 170 disposed between reagent
reservoirs 172 and the reaction compartment. The reagent delivery
system can define a plurality of compartments for holding fluid,
including the reagent reservoirs, the reaction compartment, a waste
reservoir 174 disposed downstream of the reaction compartment, and
channels 176 extending between and/or defining these structures. In
some examples, the reagent delivery system can be disposed on a
planar substrate, with microfluidic (or larger) passages for
holding and carrying fluid formed on and/or above the planar
substrate by a fluidics layer abutted to the substrate.
[0076] Nanowire array 146 can include nanowire assemblies each
coupled to nucleic acids corresponding to a template 178
base-paired with a primer 180. In operation, the nucleotide
addition reagents 156-162 can be individually dispensed to the
reaction compartment. Before, during, and/or after each addition,
the detector can measure the conductance of each nanowire assembly,
allowing a determination of whether or not each nucleotide was
attached to the primer based on the sequence of the template, and,
if added, how many subunits were attached.
[0077] FIG. 7 shows an exemplary method 200 of sequencing nucleic
acid templates, which can be performed with the sequencing system
shown in FIG. 6. The method can include a step of providing a
nanowire assembly, shown at 202. The nanowire assembly can include
a nanowire, a template, and a primer for the template. The nanowire
assembly can be part of an array of assemblies. The method can
include a step of contacting the nanowire assembly with a
nucleotide addition reagent, shown at 204. The nucleotide addition
reagent can include, for example, a polymerase and nucleoside
triphosphate. The method can include a step of measuring, shown at
206. Measuring can detect an electrical characteristic of each
nanowire assembly before, during, and/or after the step of
contacting to determine whether or not one or more nucleotides were
attached (covalently) to each nanowire assembly. The steps of
contacting and measuring can be repeated for the other nucleotide
reagents (or only a subset thereof), shown at 208. In some
examples, the steps can be repeated until each of the four
different nucleotide reagents (or a suitable subset thereof, such
as two different nucleotide reagents corresponding to two known
single nucleotide polymorphisms) have separately contacted the
nanowire assemblies. If only a single nucleotide position of the
template, immediately adjacent the end of the primer, is of
interest, the method can be terminated, shown at 210. However, if
the controller determines that the template can and/or should be
sequenced more, to obtain sequence information about additional
nucleotide positions in the template, the steps of contacting,
measuring, and repeating can be performed again, shown at 212,
during one more additional cycles.
[0078] FIG. 8 shows exemplary data that may be obtained using
method 200 of FIG. 7. Each of the four dNTPs (including polymerase)
are dispensed stepwise, shown at 220, to a pair of nanowire
assemblies 222. Graph 224 plots each nucleotide reagent dispensed,
shown at 226, against the conductance measured after each addition,
shown at 228. An increase in conductance during and after
contacting with a particular nucleotide signifies a partner
nucleotide, complementary to the particular nucleotide, at the
corresponding position in the template strand. The size of the
increase signifies how many nucleotide subunits were attached, with
the increase being generally proportional to the number of
nucleotide subunits attached to each primer molecule of a nanowire
assembly. Accordingly, the upper plot, shown at 230, indicates
covalent addition of AGGTTCAA, and the lower plot, shown at 232,
indicates covalent addition of GAGTCCA (and thus the presence of
complementary sequences at corresponding regions of the
template).
Example 4
Nanowire-Based Analysis Utilizing Ligation
[0079] This example describes nucleic acid analysis using ligation
of nucleic acids associated with nanowires; see FIG. 9.
[0080] Humans have substantially identical genomes. Accordingly,
positions of sequence variation within the population (e.g.,
single-nucleotide polymorphisms) can be utilized to identify
individuals or lineages (e.g., for forensic purposes), to diagnose
genetic diseases or conditions, and/or to predict responses to
treatment regimens (e.g., to facilitate selection of drugs), among
others. Ligation-based analysis can be suitable for sequencing
individual positions of sequence variation in the population. In
particular, ligation-based analysis can utilize the largely known
sequence of a nucleic acid analyte to generate probes that allow
sequencing of nucleotide positions of known variation among
individuals of a population.
[0081] FIG. 9 shows a flowchart 250 for a method of nanowire-based
sequencing using ligation, and exemplary data that may be obtained
using the method.
[0082] Nanowire assemblies 252, 254 can be provided. The assemblies
can include a nanowire 256 and a different probe 258, 260 coupled
to each nanowire. The probes can include a region of nucleotide
sequence variation in the human population. In particular, the
probes (or a region thereof) can differ by only one (or a few)
nucleotides, with each probe corresponding to a different sequence
version of a polymorphism in the population. Furthermore, the
sequence differences between the probes can be disposed at or near
the end of the probes, so that any nonpairing of the probe with a
template will be pronounced at the end of probe.
[0083] A template 262 having a polymorphic nucleotide 264 (in the
population) can contact, shown at 266, the nanowire assemblies. The
template can base pair with each of the probes to form nucleic acid
duplexes 268, 270. However, probe 258 can be partially unpaired in
duplex 268, shown at 272, because this probe does not form a base
pair with the polymorphic nucleotide. Probe 260 is fully paired
with the template in duplex 270, shown at 274, because this probe
does form a base pair with the polymorphic nucleotide.
[0084] An extension nucleic acid 276 and a ligase enzyme can
contact, shown at 278, the nanowire assemblies. The extension
nucleic acid can be a (5'-phosphorylated) oligonucleotide
configured to pair with the template in a position adjoining each
probe. Accordingly, apposed ends of the extension nucleic acid and
the probe can be joined by the ligase enzyme, shown at 280, to
produce a ligation product 282 that is an extended version of probe
260 in assembly 283. However, ends of the extension nucleic acid
and probe 258 are not substantially apposed for efficient ligation
in assembly 284.
[0085] Nucleic acid duplexes can be disrupted, shown at 285. This
disruption can leave original probe 258 and ligation product 282
coupled to their respective nanowires, while releasing the template
from both nanowires, and the (unligated) extension nucleic acid
from only the upper nanowire.
[0086] Conduction of the nanowires can be tested before and after
the series of operations, shown at 286 and 288, respectively.
Exemplary data that may be obtained is plotted in a graph 290.
Probe 258 was not lengthened by ligation, so conductance of its
associated nanowire is not changed, shown at 292. Probe 260 was
extended by ligation, so conductance of its associated nanowire is
increased, shown at 294.
[0087] The method presented above can be modified in various ways.
For example, a single probe can be used sequentially with different
extensions. In particular, the probe can hybridize with the
template adjacent the polymorphic nucleotide, and then different
potential extension substrates, which hybridize to different
versions of the polymorphic nucleotide can be added separately, and
tested for their ability to be ligated to the probe.
[0088] Further illustrative discussion of ligation conditions and
other aspects of ligation are described, for example, in U.S. Pat.
No. 6,511,810 (such as at column 17, line 58, to column 18, line
47), which is incorporated herein by reference
Example 5
Nanowire-Based Analysis with Cleavage
[0089] This example describes an exemplary nanowire-based system
using selective cleavage of nucleic acids with a nuclease; see FIG.
10.
[0090] Nanowire assemblies 300, 302 including distinct nucleic acid
duplexes 304, 306 can be distinguished by differential cleavage of
the duplexes, shown at 308, 310, using a nuclease. The nuclease can
selectively cut duplexes according to primary sequence, base-pair
mismatches, unpaired ends, presence of a duplex, absence of a
duplex, and/or the like. Differential cleavage of probes 312, 314
can be detected by measuring conductance of the nanowire assemblies
with or without a separate duplex disruption step. In some
examples, appropriate selection of probes can allow cleavage to be
detected as a cleavage-induced destabilization of the duplex, as
shown in the present illustration.
Example 6
Exemplary Coupling of Nucleic Acids to Nanowires
[0091] This example describes an exemplary approach for coupling
nucleic acids to nanowires at two or more sites along each nucleic
acid; see FIG. 11.
[0092] Each nucleic acid (i.e., an analyte and/or a probe) can be
coupled to a nanowire at one or more positions along the nucleic
acid. In some examples, the nucleic acid can be coupled at two or
more spaced sites (i.e., separated by one or more nucleotides of
the nucleic acid). Coupling at multiple sites can constrain the
nucleic acid to an orientation that is more parallel to the
nanowire than coupling at a single site. Accordingly, the use of
multiple coupling sites can position the nucleic acid closer to the
nanowire, with less variation in spacing from the nanowire for
different regions of the nucleic acid. Placing the nucleic acid
closer to the nanowire, and in a more constrained configuration,
can increase the sensitivity with which changes in an electrical
characteristic can be measured, and also can reduce the variation
in the changes measured with respect to different regions of the
nucleic acid. As a result, the use of multiple coupling sites per
nucleic acid molecule can allow a number of advantages over
single-site coupling, such as (1) more sequence information (i.e.,
longer reads) for each analyte, (2) sequence analysis with fewer
analyte molecules per nanowire, and/or (3) more consistent sequence
analysis of analytes, among others.
[0093] FIG. 11 shows an exemplary nanowire assembly 350 during
coupling of molecules of a nucleic acid 352 (an analyte and/or a
probe) to a nanowire 354 of the assembly at two or more sites
(e.g., sites 356, 358) along the nucleic acid. Each coupling site
can be formed by a specific binding pair 360 (see Section III
above). For example, nanowire 354 can be connected to a first
binding member 362, and nucleic acid 352 can be connected to a
second binding member 364 that binds specifically to the first
binding member. Each molecule of the nucleic acid can be connected
to (or integrally include) two or more moieties of the second
binding member, to provide two or more binding sites along the
nucleic acid. The moieties can be spaced from one another, for
example, disposed generally toward opposing ends of the nucleic
acid, to facilitate tethering two or more distinct regions of the
nucleic acid to the nanowire. Exemplary first and second binding
members can include, respectively, (1) streptavidin and biotin, (2)
biotin and streptavidin, and/or (3) complementary nucleic acids,
among others. If base-pairing is used to couple the nucleic acid to
the nanowire at two or more spaced sites, spaced regions of the
nucleic acid itself can be used for base-pairing interaction. A
base-paired partner disposed at one or more of the spaced regions
(and connected more directly to the nanowire) also can serve as a
probe/primer, or a distinct probe/primer can be hybridized to the
nucleic acid.
[0094] The disclosure set forth above may encompass multiple
distinct inventions with independent utility. Although each of
these inventions has been disclosed in its preferred form(s), the
specific embodiments thereof as disclosed and illustrated herein
are not to be considered in a limiting sense, because numerous
variations are possible. The subject matter of the inventions
includes all novel and nonobvious combinations and subcombinations
of the various elements, features, functions, and/or properties
disclosed herein. The following claims particularly point out
certain combinations and subcombinations regarded as novel and
nonobvious. Inventions embodied in other combinations and
subcombinations of features, functions, elements, and/or properties
may be claimed in applications claiming priority from this or a
related application. Such claims, whether directed to a different
invention or to the same invention, and whether broader, narrower,
equal, or different in scope to the original claims, also are
regarded as included within the subject matter of the inventions of
the present disclosure.
* * * * *