WO2012061726A2

WO2012061726A2 - Compositions and methods for sequencing dna

Info

Publication number: WO2012061726A2
Application number: PCT/US2011/059364
Authority: WO
Inventors: Carlos H. Mastrangelo; Renny E. Fernandez
Original assignee: University Of Utah Research Foundation
Priority date: 2010-11-05
Filing date: 2011-11-04
Publication date: 2012-05-10
Also published as: WO2012061726A3

Abstract

Provided are compositions and methods for sequencing a target polynucleotide that has been used to generate an elongated polynucleotide having a plurality of segments, each segment corresponding to one of the nucleic acid residues of the target polynucleotide and having a sequence motif. The composition includes a first oligonucleotide for specifically hybridizing to any segment of the elongated polynucleotide having the first sequence motif, a second oligonucleotide for specifically hybridizing to any segment of the elongated polynucleotide having the second sequence motif, a third oligonucleotide for specifically hybridizing to any segment of the elongated polynucleotide having the third sequence motif; and a fourth oligonucleotide for specifically hybridizing to any segment of the elongated polynucleotide having the fourth sequence motif. The first, second, third, and fourth oligonucleotides each independently are complexed with a first, second, third, and fourth nanoparticle, respectively. The nanoparticles are distinguishable from one another using scanning electron microscopy.

Description

COMPOSITIONS AND METHODS FOR SEQUENCING DNA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 61/456,442, filed November 5, 2010, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant ECCS-0924808 awarded by the National Science Foundation (NSF), and grant 7R21 HG004129-04 awarded by the National Institutes for Health (NIH). The Government has certain rights in this invention.

FIELD

[0003] This disclosure relates to compositions and methods for sequencing polynucleotides.

BACKGROUND

[0004] Various techniques have been developed for the sequence-specific detection of DNA. Many of the techniques involve short tagged DNA oligonucleotides selectively hybridized to longer target polynucleotide DNA molecules. Several tagging methods for the short oligonucleotides have been used, including optical fluorescence with molecular probes. However, fluorescent probes have limitations such as poor spatial resolution, quenching, photobleaching, short fluorescent lifetimes, and optical interference.

[0005] Nanoparticles have been used in some tagging methods. Nanometer-sized particles can be conjugated to short oligonucleotides, which can be hybridized to segments of longer target polynucleotide DNA molecules. However, the large size of nanoparticles has prevented their use in sequencing full-length target polynucleotides.

SUMMARY

[0006] In some aspects, this disclosure provides compositions for sequencing a target polynucleotide that has been used to generate an elongated polynucleotide having a plurality of segments, each segment corresponding to one of the nucleic acid residues of the target polynucleotide and having a sequence motif, wherein each segment corresponding to an adenine has a first sequence motif, each segment corresponding to a guanine has a second sequence motif, each segment corresponding to a cytosine has a third sequence motif, and each segment corresponding to a thymine has a fourth sequence motif, the composition comprising a first oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the first sequence motif, wherein the first oligonucleotide is complexed with a first nanoparticle; a second oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the second sequence motif, wherein the second oligonucleotide is complexed with a second nanoparticle; a third oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the third sequence motif, wherein the third oligonucleotide is complexed with a third nanoparticle; and a fourth oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the fourth sequence motif, wherein the fourth oligonucleotide is complexed with a fourth nanoparticle, wherein the first, second, third, and fourth nanoparticles are distinguishable from one another using a scanning electron microscope.

[0007] In some aspects, this disclosure provides methods of sequencing a target polynucleotide, the method comprising generating an elongated polynucleotide comprising a plurality of segments, each segment corresponding to one of the nucleic acid residues of the target polynucleotide and having a sequence motif, wherein each segment corresponding to an adenine has a first sequence motif, each segment corresponding to a guanine has a second sequence motif, each segment corresponding to a cytosine has a third sequence motif, and each segment corresponding to a thymine has a fourth sequence motif, and wherein the sequence of the segments corresponds to the nucleotide sequence of the target polynucleotide; attaching one end of the elongated polynucleotide to a surface; mixing the elongated polynucleotide with a composition comprising a first oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the first sequence motif, wherein the first oligonucleotide is complexed with a first nanoparticle; a second oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the second sequence motif, wherein the second oligonucleotide is complexed with a second nanoparticle; a third oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the third sequence motif, wherein the third oligonucleotide is complexed with a third nanoparticle; and a fourth oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the fourth sequence motif, wherein the fourth oligonucleotide is complexed with a fourth nanoparticle; whereupon the first, second, third, and fourth oligonucleotides hybridize to the first, second, third, and fourth sequence motifs of the single- stranded elongated polynucleotide, respectively; imaging the nanoparticles using a scanning electron microscope (SEM); and determining the sequence of the target polynucleotide based on the sequence of the nanoparticles in the image.

[0008] This disclosure provides other aspects and embodiments that will be apparent in light of the following detailed description and accompanying Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 is a schematic diagram showing an exemplary method of sequencing a target polynucleotide.

[0010] Figure 2 is a schematic diagram showing a polynucleotide immobilized to a solid support and hybridized to a plurality of nanoparticle-oligonucleotide complexes.

[0011] Figure 3 is a pair of SEM images showing a plurality of polynucleotides each hybridized to one nanoparticle-oligonucleotide complex, where the nanoparticles in the complexes each are all substantially the same size (-30 nm in diameter).

[0012] Figure 4 is a schematic diagram of a single polynucleotide hybridized to (a) a plurality of biotinylated oligonucleotides, and (b) a plurality of nanoparticle-oligonucleotide complexes, where the nanoparticles in the complexes are all of substantially the same size.

[0013] Figure 5 is a series of SEM images that each show a single polynucleotide hybridized to a plurality of nanoparticle-oligonucleotide complexes, where the nanoparticles in the complexes are all substantially the same size.

[0014] Figure 6 is a schematic diagram showing a polynucleotide hybridized to several different variations of nanoparticle-oligonucleotide complexes.

[0015] Figure 7 is an image showing the mobility of three different nanoparticle- oligonucleotide complexes through an agarose gel. [0016] Figure 8 is a series of SEM images, each showing a single polynucleotide hybridized to a plurality of nanoparticle-oligonucleotide complexes. The nanoparticles are 2 different sizes.

[0017] Figure 9 is a series of SEM images, each showing a single polynucleotide hybridized to a plurality of nanoparticle-oligonucleotide complexes. The nanoparticles are 3 different sizes.

[0018] Figure 10 is a series of SEM images, each showing a single polynucleotide hybridized to a plurality of nanoparticle-oligonucleotide complexes. The nanoparticles are 4 different sizes.

DETAILED DESCRIPTION

[0019] This disclosure provides compositions and methods for sequencing polynucleotides. These compositions and methods include nanoparticle-oligonucleotide complexes, which may be hybridized to segments of DNA and imaged with a scanning electron microscope (SEM).

[0020] Figure 1 is a schematic diagram showing an exemplary method of sequencing a target polynucleotide, which includes one or more of the following steps: generating an elongated polynucleotide from the target polynucleotide, attaching the elongated polynucleotide to a surface, denaturing the elongated polynucleotide, hybridizing the elongated polynucleotide to a plurality of nanoparticle-oligonucleotide complexes, and imaging the nanoparticles using an SEM.

[0021] A target polynucleotide first may be converted to an elongated polynucleotide comprising a plurality of segments, where each segment corresponds to one of the nucleic acid residues of the target polynucleotide and includes a sequence motif. For example, each segment corresponding to an adenine may include a first sequence motif, each segment corresponding to a guanine may include a second sequence motif, each segment corresponding to a cytosine may include a third sequence motif, and each segment corresponding to a thymine may include a fourth sequence motif. The first, second, third, and fourth sequence motifs each may comprise a different DNA sequence. Each sequence motif may be between about 50 and about 300 nucleotides. For example, each sequence motif may be at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, etc. Similarly, each sequence motif may be less than about 300 nucleotides, less than about 250 nucleotides, less than about 200 nucleotides, or less than about 150 nucleotides. The segments may be immediately adjacent to each other in the elongated polynucleotide, or there may be one or more nucleotides between the segments. Either with or without nucleotides in between adjacent segments, the distance between the beginnings of adjacent segments may be greater than or equal to the diameter of the largest nanoparticle. For example, if nanoparticles of 30-nm diameter are used, then each segment may be 100 bp in length and immediately adjacent to the next 100-bp segment. As such, the distance between the beginning of adjacent segments would be approximately 35 nm (100 bp of DNA is approximately 35 nm in length along the longitudinal axis) and would allow for a 30-nm diameter nanoparticle to fit. As another, and more conservative, example, each segment may be 100 bp in length with a 100 bp gap between it and the next adjacent 100-bp segment. As such, the distance between the beginnings of adjacent segments would be approximately 70 nm (200 bp of DNA is approximately 70 nm in length along the longitudinal axis). The order of the segments may correspond to the nucleotide sequence of target polynucleotide. Methods for generating an elongated polynucleotide from a target polynucleotide are described in U.S. Patent No. 6,723,513, U.S. Patent Application Publication Number 20090047744, McNally et al. (Nano Letters 2010, 10, 2237-2244), and Soni et al. (Clinical Chemistry 2007, 53, 1996-2001 ), each of which are incorporated herein by reference for all purposes. Kits for generating an elongated polynucleotide from a target polynucleotide are commercially available from, for example, Lingvitae AS (Oslo, Norway). Suitably, the elongated polynucleotide is generated as a linear dsDNA molecule with a 1 -nucleotide overhang. The elongated polynucleotide may be about 5,000 to about 50,000 nucleotides or base pairs in length. For example, the elongated polynucleotide may be at least about 5,000, at least about 6,000, or at least about 7,000 nucleotides or base pairs in length. The elongated polynucleotide may be less than about 50,000, less than about 40,000, or less than about 30,000 nucleotides or base pairs in length. The target polynucleotide may be about 50 to about 300 base pairs in length. For example, the target polynucleotide may be at least about 50, at least about 60, or at least about 70 base pairs in length. The target polynucleotide may be less than about 300, less than about 250, or less than about 200 base pairs in length. For longer sequences of DNA to be sequenced, they may be first cut by, for example, a nuclease to generate smaller pieces of DNA of about 50 to about 300 base pairs in length.

[0022] The elongated polynucleotide may be attached to a surface. Polynucleotide attachment may be by any suitable means known in the art, or hereinafter devised. For example, a polynucleotide modified with a phosphoramidite may be attached to a Si0₂ surface modified with mercaptopropyltrimethoxysilane (MPTMS), as described in the examples. As another example, a polynucleotide modified with a thiol group may be attached to a gold surface. The elongated polynucleotide may be directly attached or tethered to a surface. Alternatively or additionally, the elongated polynucleotide may be indirectly attached via a linker. For example, the elongated polynucleotide may be ligated to a short oligonucleotide linker that is attached or tethered to a surface. As described above, the elongated polynucleotide may be generated as a linear dsDNA molecule with a 1-nucleotide overhang. As such, for example, the elongated polynucleotide may be mixed with four different types of oligonucleotide linkers, each type of oligonucleotide linker independently having a G, C, T, or A overhang on one end and a phosphoramidite at the other end, to facilitate binding to a Si0₂ surface modified with MPTMS. The elongated polynucleotide may anneal to just one of the four oligonucleotide linkers, as directed by the complementary 1-nucleotide overhang. Ligase may be used to ensure complete attachment of the elongated polynucleotide to the oligonucleotide linker, thereby attaching the elongated polynucleotide to the surface. Alternatively, the 1 nucleotide overhang can be filled in to form a blunt end. Such filling-in reactions may be completed with a kit commercially available from, for example, New England Biolabs (QUICK LIGATION KIT™, Ipswich, MA). The resulting blunt-ended linear dsDNA elongated polynucleotide may be blunt-end ligated to a blunt-ended short oligonucleotide linker that is modified with phosphoramidite at the other end, to facilitate binding to a Si0₂ surface modified with MPTMS. Attachment of the elongated polynucleotide to a surface may immobilize the elongated polynucleotide to aid in hybridization of oligonucleotide-nanoparticle complexes and subsequent imaging of the nanoparticles.

[0023] The elongated polynucleotide may be generated from the target polynucleotide in double-stranded DNA (dsDNA) from. As such, the elongated polynucleotide may be denatured, so as to separate it from its complementary DNA strand and form a single-stranded DNA (ssDNA) elongated polynucleotide to facilitate hybridization of nanoparticle-oligonucleotide complexes. The dsDNA elongated polynucleotide may be denatured to form a ssDNA elongated polynucleotide before or after its attachment to a surface. Suitably, the dsDNA elongated polynucleotide may be denatured to form a ssDNA elongated polynucleotide after attachment to a surface.

[0024] A composition for sequencing the target polynucleotide may be provided, comprising: a first oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the first sequence motif, wherein the first oligonucleotide is complexed with a first nanoparticle, a second oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the second sequence motif, wherein the second oligonucleotide is complexed with a second nanoparticle, a third oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the third sequence motif, wherein the third oligonucleotide is complexed with a third nanoparticle, and a fourth oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the fourth sequence motif, wherein the fourth oligonucleotide is complexed with a fourth nanoparticle.

[0025] The oligonucleotides each may specifically hybridize to some or all of the corresponding segment. For example, the oligonucleotides each may include a sequence complementary to at least a portion of the sequence of the corresponding sequence motif. Each oligonucleotide may be between about 40 nucleotides and about 200 nucleotides in length. For example, among other lengths, each oligonucleotide may be at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, or at least about 80 nucleotides in length. Among other lengths, each oligonucleotide may be less than about 200 nucleotides, less than about 180 nucleotides, less than about 170 nucleotides, less than about 160 nucleotides, or less than about 150 nucleotides in length.

[0026] The first, second, third and fourth nanoparticles may be selected so as to be distinguishable from one another using SEM. For example, the different nanoparticles may be distinguishable by size, elemental composition, or a combination thereof. The nanoparticles each may have a different diameter. The diameter of each nanoparticle may be between about 5 nm and about 200 nm. However, it is noted that as the size of the nanoparticles increases, stickiness of the nanoparticles and difficulty in SEM increase. Suitably, the diameter of each nanoparticle may be between about 5 nm and about 50 nm. For example, among other sizes, the diameter of each nanoparticle may be at least about 5 nm, at least about 10 nm, or at least about 15 nm. Among other sizes, the diameter of each nanoparticle may be less than about 50 nm, less than about 45 nm, less than about 40 nm, or less than about 35 nm. To be optimally distinguishable from each other using SEM, the difference in diameters between the various nanoparticles may be at least about 4 nm, or at least about 5 nm. The nanoparticles each may be comprised of a different elemental composition. For example, a nanoparticle may be comprised of at least one of silver, gold, iron oxide, and titantium dioxide. Using different sizes, elemental composition, or a combination thereof, at least 4 different nanoparticles may be used. While 4 different nanoparticles are suitably used for methods of sequencing a target polynucleotide as disclosed herein, it is envisioned, that at least 4, and up to any number of different nanoparticles may be used.

[0027] The nanoparticle in each nanoparticle-oligonucleotide complex may be attached to or complexed with the corresponding oligonucleotide by any suitable means. For example, the nanoparticle may be attached to the oligonucleotide by streptavidin-avidin interaction, such as by attaching biotin to one end of the oligonucleotide, and by conjugating the nanoparticle to streptavidin. Biotin-modified oligonucleotides are commercially available from, for example, Integrated DNA Technologies (Coralville, IA). Streptavidin-conjugated nanoparticles are commercially available from, for example, Nanopartz (Loveland, CO).

[0028] The composition for sequencing the target polynucleotide may be mixed with or added to the elongated polynucleotide. For example, the nanoparticle-oligonucleotide complexes may be mixed with the elongated polynucleotide, under stringent hybridization conditions, after the elongated polynucleotide has been attached to a surface and denatured to separate the elongated polynucleotide from its complementary DNA strand and form a ssDNA elongated DNA molecule. The nanoparticle-oligonucleotide complexes may be allowed to hybridize with the elongated polynucleotide according to well-known methods, after which the nanoparticles may be imaged using an SEM. The resulting image may show a string of nanoparticles corresponding to the underlying sequence of the target polynucleotide. Since the nanoparticles are much more massive than the sequence motifs, the sequence motif order can be directly observed using SEM. The magnification may be down to about 100 nm to about 500 nm to distinguish the nanoparticles and sequence the polynucleotide.

[0029] Compositions

[0030] This disclosure provides compositions for sequencing target polynucleotides, and specifically, polynucleotides that have been used to generate an elongated polynucleotide having a plurality of segments, each segment corresponding to one of the nucleic acid residues of the target polynucleotide and having a sequence motif, as described above. The composition comprises a first oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the first sequence motif, wherein the first oligonucleotide is attached to a first nanoparticle, a second oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the second sequence motif, wherein the second oligonucleotide is attached to a second nanoparticle, a third oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the third sequence motif, wherein the third oligonucleotide is attached to a third nanoparticle, and a fourth oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the fourth sequence motif, wherein the fourth oligonucleotide is attached to a fourth nanoparticle. The first, second, third, and fourth nanoparticles are distinguishable from one another using a scanning electron microscope.

[0031] In another aspect, this disclosure provides kits for sequencing target polynucleotides. These kits may comprise a first container containing the first, second, third, and fourth oligonucleotides attached to first, second, third, and fourth nanoparticle, respectively, as described above. The kits further may comprise a second container containing a composition for generating the elongated polynucleotide from the target polynucleotide. As detailed above, methods for generating an elongated polynucleotide from a target polynucleotide are described in U.S. Patent No. 6,723,513, U.S. Patent Application Publication Number 20090047744, McNally et al. (Nano Letters 2010, 10, 2237-2244), and Soni et al. (Clinical Chemistry 2007, 53, 1996-2001 ), each of which are incorporated herein by reference for all purposes. Kits for generating an elongated polynucleotide from a target polynucleotide are commercially available from, for example, Lingvitae AS (Oslo, Norway). Suitably, the elongated polynucleotide is generated as a linear dsDNA molecule with a 1 -nucleotide overhang.

[0032] Methods

[0033] Once the segments of the elongated polynucleotide are hybridized to nanoparticle- oligonucleotide complexes, the different nanoparticles may be imaged with SEM. The Examples demonstrate the use of this technique for the direct sequencing of segments in single DNA molecules consisting of four 100-nt segments hybridized to different types of nanoparticle- oligonucleotide complexes.

[0034] In an aspect, provided is a method of sequencing a target polynucleotide, the method comprising generating an elongated polynucleotide comprising a plurality of segments, each segment corresponding to one of the nucleic acid residues of the target polynucleotide and having a sequence motif, as described above. That is, each segment corresponding to an adenine has a first sequence motif, each segment corresponding to a guanine has a second sequence motif, each segment corresponding to a cytosine has a third sequence motif, and each segment corresponding to a thymine has a fourth sequence motif, and wherein the sequence of the segments corresponds to the nucleotide sequence of the target polynucleotide. The methods further comprise attaching one end of the elongated polynucleotide to a surface, as described above. The elongated polynucleotide may be double-stranded or single-stranded. If the elongated polynucleotide is doubled-stranded DNA (dsDNA), it may be first denatured to form a single-stranded DNA (ssDNA) elongated polynucleotide for the nanoparticle- oligonucleotide complexes to hybridize. The methods further comprise mixing the elongated polynucleotide with a composition comprising the first, second, third, and fourth oligonucleotides attached to first, second, third, and fourth nanoparticles, respectively, as described above. The first, second, third, and fourth oligonucleotides hybridize to the first, second, third, and fourth sequence motifs of the single-stranded elongated polynucleotide, respectively. The methods further comprise; imaging the nanoparticles using a scanning electron microscope (SEM), and determining the sequence of the target polynucleotide based on the sequence of the nanoparticles in the image. Some steps of the method are shown schematically in Figure 1.

[0035] SEM, and not transmission electron microscopy (TEM), is suitable for imaging the nanoparticles as described herein. TEM imaging can indeed be used to image individual atoms, but it requires very tedious and meticulous sample prep and each image requires time to refocus. Further, the field of view using TEM is very small (approximately 5 nm on each side) and unsuitable for efficient sequencing of polynucleotides. TEM is not suitable for high throughput scanning, whereas SEM is suitable for high throughput screening. Each of the nanoparticles described herein may contain between about 5,000-10,000 atoms of gold or other metal atoms. Because these nanoparticles are so big (about 5-50 nm in diameter instead of 0.4 nm for single atoms), they easily can be seen with an existing SEM tool.

[0036] In particular, the realization of four-particle encoded vectors, which are clearly imaged by conventional SEM techniques, is of high technological relevance because of its direct applicability to sequencing. For example, if the nanoparticle tagged fragments are ordered on a substrate, an information packing density of 1 kb/μηη² could be reached, with a capacity of 100 Gb/cm². With SEM scanning rates of 2 x 2 μηη² in 20 ms, the substrate genomic information could be read out at a rate of 720 Mb/hour, about two times faster than Life Technologies state- of-the-art SOLIDTM system (-300 Mb/hour). Due to the information transfer from an organic molecule to essentially a nanoparticle chain, the methods described herein could produce a solid-state memory imprint of a genome. Further, SEM may be completely automated.

[0037] The technology offers a robust alternative to the fluorophore based approach and, to a large extent, circumvents the problems associated with fluorescence (quenching, low resolution etc.).

[0038] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise noted. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to illustrate aspects and embodiments of the disclosure and does not limit the scope of the claims.

EXAMPLES

Example 1 : Materials and Methods [0039] Materials

[0040] The vector pET-3a (SEQ ID NO: 1 ) was purchased from New England Biolabs (Ipswich, MA). Custom modified (for example, biotinylated or phosphoramidite-modified) single- stranded DNA (ssDNA) oligonucleotides were ordered from Integrated DNA Technologies (Coralville, IA). Nanoparticles were purchased from Nanopartz (Loveland, CO). All the chemicals used were of molecular biology grade and were purchased from Sigma-Aldrich (St. Louis, MO). An FEI NanoNova Scanning Electron Microscope (SEM; FEI, Hillsboro, OR), having a resolution of 2 nm, was used to image the samples. The SEM was operated in vacuum mode, and a low-voltage high-contrast detector (VCD) was used.

[0041] Immobilization of DNA Polynucleotides

[0042] DNA polynucleotides were immobilized to a surface to produce long linear DNA structures that were easy to read by combing techniques. A short dsDNA oligonucleotide linker modified at the 5'-end with phosphoramidite (ACRYDITE™, Integrated DNA Technologies, Coralville, IA) was used. A longer dsDNA polynucleotide was ligated to the other end of the phosphoramidite-modified oligonucleotide linker, to form a phosphoramidite-modified long dsDNA polynucleotide.

[0043] Oxidized silicon (Si0₂) wafers were treated with 5 mM mercaptopropyltnmethoxysilane (MPTMS) for 48 hours to form a monolayer of MPTMS with exposed thiol end groups (SH). The surface was then washed with deionized water, acetone, and methanol. A concentration of 5 mM MPTMS was used to reduce the potential for a disordered and disoriented surface with less thiol headgroups on the uppermost surface. The resulting MPTMS layer with a thiol-terminated surface served as a coupling agent between the substrate surface and the oligonucleotide linker. The exposed thiol end groups of the MPTMS monolayers were mixed with the phosphoramidite-modified long dsDNA polynucleotide, which was thereby immobilized to the Si0₂ surface.

[0044] Attachment of the polynucleotide to the surface may be before or after denaturation and hybridization of nanoparticle-oligonucleotide complexes, as detailed in the Examples below.

[0045] Formation of Nanoparticle-Oligonucleotide Complexes

[0046] Biotinylated single-stranded (ssDNA) oligonucleotides complementary to specific segments of the long DNA polynucleotide (for example, to segments of the linearized pET-3a polynucleotide) were selected. The oligonucleotides were designed to counter self looping and non-specific binding according to known methods. The oligonucleotides were attached to steptavidin-conjugated gold nanoparticles of various sizes as follows: 5, 10, 20, or 30 nm in diameter (designated P1 , P2, P3, and P4, respectively). A streptavidin-conjugated nanoparticle was attached to one end of the selected biotinylated oligonucleotide to form nanoparticle- oligonucleotide complexes. Each oligonucleotide had a different-sized gold nanoparticle attached to it. The complexes were formed either before or after the oligonucleotide(s) was hybridized to the DNA polynucleotide, depending on the application as discussed in the examples below. [0047] Hybridization of the Nanoparticle-Oligonucleotide Complexes to the DNA Polynucleotide and Imaging of Nanoparticles

[0048] The long dsDNA polynucleotide was denatured at a high temperature (95°C), and the nanoparticle-oligonucleotide complexes were specifically hybridized to segments of the DNA polynucleotide. Figure 2 shows a schematic representation of examples of some DNA polynucleotides immobilized to a surface and hybridized to nanoparticle-oligonucleotide complexes used in this study. This was followed by scanning electron microscopy (SEM) imaging of the resulting DNA polynucleotide hybridized to the nanoparticle-oligonucleotide complexes. The nanoparticles (and thus the order of the nanoparticles) were imaged. The experiments were carried out on both surface-bound and free-solution DNA polynucleotides.

Example 2: Single Polynucleotide Tagged with a Single Nanoparticle

[0049] In order to determine the feasibility of the nanoparticle tagging scheme, various oligonucleotides that were all complexed with nanoparticles of substantially uniform size were hybridized to DNA polynucleotides. First a single motif or segment of the DNA polynucleotide was tagged with a single nanoparticle.

[0050] To attach the polynucleotide to a surface, ACRYDITE™-modified 78-bp dsDNA was first ligated with a 200 bp dsDNA polynucleotide. The ligated DNA polynucleotide (278 bp) was then immobilized on a MPTMS-treated Si0₂ surface, as described in Example 1. The immobilized DNA polynucleotide was next heated at 95°C for denaturation to generate a single- stranded 278 nt polynucleotide. A biotinylated ssDNA probe (1 12 nt) was next hybridized to a single motif or segment in the immobilized DNA. Subsequently, the samples were immersed in a solution of streptavidin-conjugated P1 nanoparticles (gold, 30 nm diameter) overnight to bind to the biotinylated ssDNA probe. The samples were next washed three times in TE buffer (pH 7.6) to flush out non-specifically bound nanoparticles. The nanoparticles selected were large enough for SEM imaging but small enough to avoid overcrowding and coagulation.

[0051] Figure 3 shows an SEM image of a MPTMS-treated Si0₂ surface after DNA immobilization and subsequent P1 (30 nm diameter) nanoparticle attachment. A single nanoparticle bound to each immobilized DNA molecule. The blank samples (with no DNA attached) did not retain nanoparticles after they were washed three times in TE buffer (pH 7.6). Example 3: Multiple Segments of a Single Polynucleotide Tagged with Multiple Homogenous Nanoparticles

[0052] Next multiple homogeneous nanoparticle-oligonucleotide complexes (with nanoparticles of substantially the same size) were hybridized to multiple segments of a single long polynucleotide as shown schematically in Figure 4. Linearized pET-3a dsDNA was first ligated to a short dsDNA oligonucleotide immobilized on the Si0₂ surface, as described in Example 1. The resulting long dsDNA polynucleotide was next denatured. Then, a set of short biotinylated ssDNA oligonucleotides, complementary to segments of the pET-3a polynucleotide, was hybridized to the long ssDNA polynucleotide bound to the surface.

[0053] Then streptavidin-conjugated gold P1 nanoparticles (30 nm diameter) were added. The nanoparticles were allowed to attach to the biotinylated oligonucleotides. Nanoparticles of the same size were hence attached to a single long DNA polynucleotide. The nanoparticles selected were large enough for SEM imaging but small enough to avoid overcrowding and coagulation.

[0054] The samples were next dried for SEM imaging. Figures 5a-b show the simultaneous tagging of three separate 100-nt segments of pET-3a, each segment separated by 100 nt, using P1 nanoparticles. Figure 5c shows the linkage of seven P1 nanoparticles to seven sequential 100 nt segments of pET-3a. These preliminary experiments indeed demonstrated the correct imaging of nanoparticle-oligonucleotide hybridization to a known number of segments in the pET-3a polynucleotide.

Example 4: Multiple Segments of a Single Polynucleotide Tagged with Multiple Heterogenous Nanoparticles

[0055] In order to link nanoparticles of different sizes to linearized pET-3a segments, a different scheme was adopted. The ssDNA oligonucleotides complementary to one of the several segments of the long polynucleotide were first linked with different nanoparticles of the specific size required for each pattern, before hybridization of the nanoparticle-oligonucleotide complexes to the DNA polynucleotide. For instance, to generate the particle index vector (2, 1 , 4), the ssDNA oligonucleotides for segments 1 , 2, and 3 were linked to nanoparticles P2, P1 , and P4, respectively. However, to generate vector (1 , 2, 4), oligonucleotides for segments 1 , 2, and 3 were linked with nanoparticles P1 , P2, and P4, respectively. [0056] The relevant short ssDNA oligonucleotides were added into aqueous dispersions of the corresponding streptavidin-conjugated gold nanoparticle. The mixture was incubated at room temperature for 24 hours. Subsequently, the aqueous solution of NaCI (5 mol/L, 50 μΙ_) was added into the mixture solution. After 24 hours, an additional 50 μΙ_ NaCI (5 mol/L) was added. After further incubation for 24 hours, the nanoparticle-oligonucleotide complexes were centrifuged for 10 min at 10,000 rpm. The precipitate was washed three times with 0.3 mol/L NaCI, 10 mmol/L phosphate buffer (pH 7.0, referred as 0.3 mol/L PBS) to remove the excess non-conjugated oligonucleotides. The nanoparticle-oligonucleotide complexes were re-solvated with nuclease-free water.

[0057] The nanoparticle-oligonucleotide complexes were then added to a solution containing linearized and denatured pET-3a for hybridization. Samples were imaged using a scanning electron microscope.

[0058] This strategy produced chains of nanoparticles attached to segments of the long pET-3a polynucleotide. The method was useful for identification of segment order, and this procedure can be utilized as a precursor in DNA sequencing applications. Several combinations of nanoparticle-oligonucleotide complexes that were used are shown schematically in Figure 6. The sequences of the oligonucleotides used are given in Table 1.

Table 1. Sequences of the ssDNA oligonucleotides used in the experiment.

SEQ ID TATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTC

NO: 6

Motif 6 TTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGT I M G

SEQ ID CAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCA NO: 7

Motif 7 AATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCA

SEQ ID CTGATGCCTCCGTGTAAGGGGGA 1 1 1 CTGTTCATGGGGGTA

NO: 8

[0059] Figure 7 is a 2% agarose gel of nanoparticle-oligonucleotide complexes. The electrophoresis was performed for 25 minutes to separate the oligonucleotides conjugated with nanoparticles. The distinct bands can be identified from the figure. Lane 2, 3, 4 are oligonucleotides with 20 nm, 10 nm, and 5 nm nanoparticles, respectively. As expected, the oligonucleotides conjugated with smaller nanoparticles migrated faster than those with the bigger nanoparticles (5 >10 >20 nm). Lane 1 was loaded with oligonucleotides conjugated with 30 nm nanoparticles; however it did not migrate in the 2% gel.

[0060] The nanoparticle-oligonucleotide complexes hybridized to the long pET-3a polynucleotide are shown in Figure 8, Figure 9, and Figure 10. Figure 8 shows SEM images of a few DNA polynucleotides hybridized with nanoparticle-oligonucleotide complexes formed using nanoparticles of two different sizes. Figure 9 shows SEM images of a few DNA polynucleotides hybridized with nanoparticle-oligonucleotide complexes formed using three different nanoparticles. Figure 10 shows SEM images of a few DNA polynucleotides hybridized with nanoparticle-oligonucleotide complexes formed using four different nanoparticles. All the conjugates shown in Figure 6 were imaged and distinguished.

[0061] With successful tagging, the resulting nanoparticle chain pattern specified by its particle index can correspond exactly to the sequence of segments in an elongated polynucleotide, and hence, also to the sequence of the target polynucleotide used to generate the elongated polynucleotide. Example 5: Sequencing of a Target Polynucleotide

[0062] A target polynucleotide of approximately 100 bp is sequenced. First, the target polynucleotide is used to generate an elongated polynucleotide. The elongated polynucleotide is generated using techniques described in U.S. Patent No. 6,723,513, U.S. Patent Application Publication Number 20090047744, McNally et al. (Nano Letters 2010, 10, 2237-2244), and Soni et al. (Clinical Chemistry 2007, 53, 1996-2001 ), and with a kit commercially available from, for example, Lingvitae AS (Oslo, Norway). The generated elongated polynucleotide is a linear dsDNA molecule with a 1-nucleotide overhang at one end. The elongated polynucleotide has a plurality of segments, each segment corresponding to one of the nucleic acid residues of the target polynucleotide and having a sequence motif, wherein each segment corresponding to an adenine has a first sequence motif, each segment corresponding to a guanine has a second sequence motif, each segment corresponding to a cytosine has a third sequence motif, and each segment corresponding to a thymine has a fourth sequence motif. Each segment is about 100 bp in length and is separated from the adjacent segment by a gap of about 100 bp in length. Thus, the elongated polynucleotide is about 20,000 bp long.

[0063] In a separate reaction, four different nanoparticle-oligonucleotide complexes are generated. A first oligonucleotide (100 nt long) is biotinylated at the 5-end and has a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the first sequence motif. A second oligonucleotide (100 nt long) is biotinylated at the 5-end and has a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the second sequence motif. A third oligonucleotide (100 nt long) is biotinylated at the 5-end and has a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the third sequence motif. A fourth oligonucleotide (100 nt long) is biotinylated at the 5- end and has a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the fourth sequence motif. The first oligonucleotide is complexed with a first streptavidin-conjugated gold nanoparticle that is 5 nm in diameter. The second oligonucleotide is complexed with a second streptavidin-conjugated gold nanoparticle that is 10 nm in diameter. The third oligonucleotide is complexed with a third streptavidin-conjugated gold nanoparticle that is 20 nm in diameter. The fourth oligonucleotide is complexed with a fourth streptavidin-conjugated gold nanoparticle that is 30 nm in diameter. The relevant short ssDNA biotinylated-oligonucleotides are added into separate aqueous dispersions of the corresponding streptavidin-conjugated gold nanoparticles. The mixture is incubated at room temperature for 24 hours. Subsequently, the aqueous solution of NaCI (5 mol/L, 50 μΙ_) is added into the mixture solution. After 24 hours, an additional 50 μΙ_ NaCI (5 mol/L) is added. After further incubation for 24 hours, the nanoparticle-oligonucleotide complexes are centrifuged for 10 min at 10,000 rpm. The precipitate is washed three times with 0.3 mol/L NaCI, 10 mmol/L phosphate buffer (pH 7.0, referred as 0.3 mol/L PBS) to remove the excess non-conjugated oligonucleotides. The nanoparticle-oligonucleotide complexes are re-solvated with nuclease- free water.

[0064] Next, the elongated polynucleotide is mixed with a composition including four different 90-bp oligonucleotides. Each 90-bp oligonucleotide has a 1 -nucleotide overhang that is T, C, G, or A. Each 90-bp oligonucleotide is also modified at the opposite end with a 5'- phosphoramidite. The elongated polynucleotide anneals with one of the 90-bp oligonucleotides, and ligase is added to the reaction to ligate the elongated polynucleotide to the oligonucleotide. Thus, a linear 20,090 bp dsDNA polynucleotide modified at the 5'-end with phosphoramidite and including the elongated polynucleotide at the other end is generated.

[0065] Oxidized silicon (Si0₂) wafers are first treated with 5 mM Mercaptopropyltnmethoxysilane (MPTMS) for 48 hours to form a monolayer of MPTMS with exposed thiol end groups (SH). The surface is then washed with deionized water, acetone, and methanol. A MPTMS layer with a thiol-terminated surface results. The exposed thiol end groups of the MPTMS monolayers are then reacted with the linear 20,0090 bp dsDNA polynucleotide modified at the 5'-end with phosphoramidite. Thus, the elongated polynucleotide is immobilized to the surface.

[0066] The elongated polynucleotide is denatured by heating at 95°C to separate the complementary strand and generate a ssDNA elongated polynucleotide. The four different nanoparticle-oligonucleotide complexes are added. The nanoparticle-oligonucleotide complexes hybridize to the corresponding segments of the elongated polynucleotide.

[0067] The nanoparticles of the elongated polynucleotide hybridized with the nanoparticle- oligonucleotide complexes are imaged with SEM. The sequence of the target polynucleotide is deduced according to the sequence of the nanoparticles in the SEM image. SEQUENCE LISTING

SEQ ID NO: 1

pET-3a polynucleotide, 4640 bp

1 TTCTCATGTT TGACAGCTTA TCATCGATAA GCTTTAATGC GGTAGTTTAT 51 CACAGTTAAA TTGCTAACGC AGTCAGGCAC CGTGTATGAA A CTAACAAT 101 GCGCTCATCG TCATCCTCGG CACCGTCACC CTGGATGCTG TAGGCATAGG 151 CTTGGTTATG CCGGTACTGC CGGGCCTCTT GCGGGATATC GTCCATTCCG 201 ACAGCATCGC CAGTCACTAT GGCGTGCTGC TAGCGCTATA TGCGTTGATG 251 CAATTTCTAT GCGCACCCGT TCTCGGAGCA CTGTCCGACC GCTTTGGCCG 301 CCGCCCAGTC CTGCTCGCTT CGCTACTTGG AGCCACTATC GACTACGCGA 351 TCATGGCGAC CACACCCGTC CTGTGGATAT CCGGATATAG TTCCTCCTTT 401 CAGCAAAAAA CCCCTCAAGA CCCGTTTAGA GGCCCCAAGG GGTTATGCTA 451 GTTATTGCTC AGCGGTGGCA GCAGCCAACT CAGCTTCCTT TCGGGCTTTG 501 TTAGCAGCCG GATCCGCGAC CCATTTGCTG TCCACCAGTC ATGCTAGCCA 551 TATGTATATC TCCTTCTTAA AGTTAAACAA AATTATTTCT AGAGGGAAAC 601 CGTTGTGGTC TCCCTATAGT GAGTCGTATT AATTTCGCGG GATCGAGATC 651 TCGATCCTCT ACGCCGGACG CATCGTGGCC GGCATCACCG GCGCCACAGG 701 TGCGGTTGCT GGCGCCTATA TCGCCGACAT CACCGATGGG GAAGATCGGG 751 CTCGCCACTT CGGGCTCATG AGCGCTTGTT TCGGCGTGGG TATGGTGGCA 801 GGCCCCGTGG CCGGGGGACT GTTGGGCGCC ATCTCCTTGC ATGCACCATT 851 CCTTGCGGCG GCGGTGCTCA ACGGCCTCAA CCTACTACTG GGCTGCTTCC 901 TAATGCAGGA GTCGCATAAG GGAGAGCGTC GACCGATGCC CTTGAGAGCC 951 TTCAACCCAG TCAGCTCCTT CCGGTGGGCG CGGGGCATGA CTATCGTCGC 1001 CGCACTTATG ACTGTCTTCT TTATCATGCA ACTCGTAGGA CAGGTGCCGG 1051 CAGCGCTCTG GGTCATTTTC GGCGAGGACC GCTTTCGCTG GAGCGCGACG 1101 ATGATCGGCC TGTCGCTTGC GGTATTCGGA ATCTTGCACG CCCTCGCTCA 1151 AGCCTTCGTC ACTGGTCCCG CCACCAAACG TTTCGGCGAG AAGCAGGCCA 1201 TTATCGCCGG CATGGCGGCC GACGCGCTGG GCTACGTCTT GCTGGCGTTC 1251 GCGACGCGAG GCTGGATGGC CTTCCCCATT ATGATTCTTC TCGCTTCCGG 1301 CGGCATCGGG ATGCCCGCGT TGCAGGCCAT GCTGTCCAGG CAGGTAGATG 1351 ACGACCATCA GGGACAGCTT CAAGGATCGC TCGCGGCTCT TACCAGCCTA 1401 ACTTCGATCA CTGGACCGCT GATCGTCACG GCGATTTATG CCGCCTCGGC 1451 GAGCACATGG AACGGGTTGG CATGGATTGT AGGCGCCGCC CTATACCTTG 1501 TCTGCCTCCC CGCGTTGCGT CGCGGTGCAT GGAGCCGGGC CACCTCGACC 1551 TGAATGGAAG CCGGCGGCAC CTCGCTAACG GATTCACCAC TCCAAGAATT 1601 GGAGCCAATC AATTCTTGCG GAGAACTGTG AATGCGCAAA CCAACCCTTG 1651 GCAGAACATA TCCATCGCGT CCGCCATCTC CAGCAGCCGC ACGCGGCGCA 1701 TCTCGGGCAG CGTTGGGTCC TGGCCACGGG TGCGCATGAT CGTGCTCCTG 1751 TCGTTGAGGA CCCGGCTAGG CTGGCGGGGT TGCCTTACTG GTTAGCAGAA 1801 TGAATCACCG ATACGCGAGC GAACGTGAAG CGACTGCTGC TGCAAAACGT 1851 CTGCGACCTG AGCAACAACA TGAATGGTCT TCGGTTTCCG TGTTTCGTAA 1901 AGTCTGGAAA CGCGGAAGTC AGCGCCCTGC ACCATTATGT TCCGGATCTG 1951 CATCGCAGGA TGCTGCTGGC TACCCTGTGG AACACCTACA TCTGTATTAA 2001 CGAAGCGCTG GCATTGACCC TGAGTGATTT TTCTCTGGTC CCGCCGCATC 2051 CATACCGCCA GTTGTTTACC CTCACAACGT TCCAGTAACC GGGCATGTTC 2101 ATCATCAGTA ACCCGTATCG TGAGCATCCT CTCTCGTTTC ATCGGTATCA 2151 TTACCCCCAT GAACAGAAAT CCCCCTTACA CGGAGGCATC AGTGACCAAA 2201 CAGGAAAAAA CCGCCCTTAA CATGGCCCGC TTTATCAGAA GCCAGACATT 2251 AACGCTTCTG GAGAAACTCA ACGAGCTGGA CGCGGATGAA CAGGCAGACA 2301 TCTGTGAATC GCTTCACGAC CACGCTGATG AGCTTTACCG CAGCTGCCTC 2351 GCGCGTTTCG GTGATGACGG TGAAAACCTC TGACACATGC AGCTCCCGGA 2401 GACGGTCACA GCTTGTCTGT AAGCGGATGC CGGGAGCAGA CAAGCCCGTC 2451 AGGGCGCGTC AGCGGGTGTT GGCGGGTGTC GGGGCGCAGC CATGACCCAG 2501 TCACGTAGCG ATAGCGGAGT GTATACTGGC TTAACTATGC GGCATCAGAG 2551 CAGATTGTAC TGAGAGTGCA CCATATATGC GGTGTGAAAT ACCGCACAGA 2601 TGCGTAAGGA GAAAATACCG CATCAGGCGC TCTTCCGCTT CCTCGCTCAC 2651 TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT 2701 CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG 2751 AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC 2801 GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA 2851 ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC 2901 CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT 2951 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC 3001 TTTCTCATAG CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC 3051 TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC 3101 CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT 3151 CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA 3201 GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG 3251 AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA 3301 AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT 3351 GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA 3401 AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA 3451 ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC 3501 TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA 3551 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA 3601 TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC 3651 GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC 3701 AATGATACCG CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA 3751 ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC 3801 GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC 3851 GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTGCA GGCATCGTGG 3901 TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA 3951 TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC 4001 CTTCGGTCCT CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC 4051 TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA 4101 AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA 4151 GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAACA CGGGATAATA 4201 CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT 4251 TCGGGGCGAA AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT 4301 GTAACCCACT CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA 4351 GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA 4401 ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA 4451 TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG 4501 AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA 4551 AAAGTGCCAC CTGACGTCTA AGAAACCATT ATTATCATGA CATTAACCTA 4601 TAAAAATAGG CGTATCACGA GGCCCTTTCG TCTTCAAGAA SEQ ID NO: 2

Motif 1 , 100 nt

CCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACG GTGCCTGACTGCGTTAGCAATTTAACTGTG

SEQ ID NO: 3

Motif 2, 100 nt

CAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCC TCTAAACGGGTCTTGAGGGGTTTTTTGCTG

SEQ ID NO: 4

Motif 3, 100 nt

AATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGA AACAAGCGCTCATGAGCCCGAAGTGGCGAG

SEQ ID NO: 5

Motif 4, 100 nt

TGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGAT TCCGAATACCGCAAGCGACAGGCCGATCAT

SEQ ID NO: 6

Motif 5, 100 nt

GGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCT ACAATCCATGCCAACCCGTTCCATGTGCTC

SEQ ID NO: 7

Motif 6, 100 nt

TTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCG CTTCACGTTCGCTCGCGTATCGGTGATTCA

SEQ ID NO: 8

Motif 7, 100 nt

AATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCG TGTAAGGGGGATTTCTGTTCATGGGGGTA

Claims

CLAIMS We claim:

1. A composition for sequencing a target polynucleotide that has been used to generate an elongated polynucleotide having a plurality of segments, each segment corresponding to one of the nucleic acid residues of the target polynucleotide and having a sequence motif, wherein each segment corresponding to an adenine has a first sequence motif, each segment corresponding to a guanine has a second sequence motif, each segment corresponding to a cytosine has a third sequence motif, and each segment corresponding to a thymine has a fourth sequence motif, the composition comprising:

a first oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the first sequence motif, wherein the first oligonucleotide is complexed with a first nanoparticle;

a second oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the second sequence motif, wherein the second oligonucleotide is complexed with a second nanoparticle;

a third oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the third sequence motif, wherein the third oligonucleotide is complexed with a third nanoparticle; and

a fourth oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the fourth sequence motif, wherein the fourth oligonucleotide is complexed with a fourth nanoparticle,

wherein the first, second, third, and fourth nanoparticles are distinguishable from one another using a scanning electron microscope.

2. The composition of claim 1 , wherein the first, second, third, and fourth nanoparticles are distinguishable by size, elemental composition, or a combination thereof.

3. The composition of claim 2, wherein the first, second, third, and fourth nanoparticles each have a different diameter, each diameter being about 5 nm to about 50 nm.

4. The composition of claim 2, wherein the first, second, third, and fourth nanoparticles each independently are comprised of at least one of silver, gold, iron oxide, and titantium dioxide.

5. The composition of claim 1 , wherein the first, second, third, and fourth oligonucleotides each independently comprise between about 40 nucleotides and about 200 nucleotides.

6. The composition of claim 1 , wherein each of the first, second, third, and fourth oligonucleotides is biotinylated; each of the first, second, third, and fourth nanoparticles is conjugated with strepatavidin; and each nanoparticle is complexed with each corresponding oligonucleotide via an interaction between the biotin and the streptavidin.

7. A kit comprising:

a first container containing the composition of claim 1 ; and

a second container containing a composition for generating the elongated polynucleotide from the target polynucleotide.

8. The kit of claim 7, wherein the elongated polynucleotide comprises between about 5,000 nucleotides and about 50,000 nucleotides or base pairs.

9. The kit of claim 7, wherein the first, second, third, and fourth sequence motifs each independently comprises between about 50 nucleotides or base pairs and about 300 nucleotides or base pairs.

10. A method of sequencing a target polynucleotide, the method comprising:

generating an elongated polynucleotide comprising a plurality of segments, each segment corresponding to one of the nucleic acid residues of the target polynucleotide and having a sequence motif, wherein each segment corresponding to an adenine has a first sequence motif, each segment corresponding to a guanine has a second sequence motif, each segment corresponding to a cytosine has a third sequence motif, and each segment corresponding to a thymine has a fourth sequence motif, and wherein the sequence of the segments corresponds to the nucleotide sequence of the target polynucleotide;

attaching one end of the elongated polynucleotide to a surface; mixing the elongated polynucleotide with a composition comprising:

a fourth oligonucleotide having a sequence for specifically hybridizing to any segment of the elongated polynucleotide having the fourth sequence motif, wherein the fourth oligonucleotide is complexed with a fourth nanoparticle;

whereupon the first, second, third, and fourth oligonucleotides hybridize to the first, second, third, and fourth sequence motifs of the elongated polynucleotide, respectively;

imaging the nanoparticles using a scanning electron microscope (SEM); and

determining the sequence of the target polynucleotide based on the sequence of the nanoparticles in the image.

11. The method of claim 10, further comprising removing unhybridized first, second, third, and fourth oligonucleotides prior to the imaging step.

12. The method of claim 10, wherein the first, second, third, and fourth nanoparticles each independently have a diameter of between about 5 nm to about 50 nm.

13. The method of claim 10, wherein the first, second, third, and fourth nanoparticles each independently are comprised of at least one of silver, gold, iron oxide, and titantium dioxide.

14. The method of claim 10, wherein the first, second, third, and fourth sequence motifs each independently comprise a sequence between about 50 nucleotides or base pairs and about 300 nucleotides or base pairs.

15. The method of claim 10, wherein the first, second, third, and fourth oligonucleotides each independently comprise a sequence of between about 40 nucleotides and about 200 nucleotides.

16. The method of claim 10, wherein the elongated polynucleotide comprises a sequence of between about 5,000 nucleotides or base pairs and about 50,000 nucleotides or base pairs.

17. The method of claim 10, wherein the generating step comprises forming the elongated polynucleotide as a double-stranded elongated polynucleotide, the attaching step comprises attaching the double-stranded elongated polynucleotide to the surface, and the method further comprises denaturing the double-stranded elongated polynucleotide to form a single-stranded elongated polynucleotide.

18. The method of claim 17, wherein the attaching step is completed before the denaturing step.

19. The method of claim 10, wherein in the attaching step, the one end of the elongated polynucleotide is attached to a surface comprising Si0₂.

20. The method of claim 19, wherein the one end of the elongated polynucleotide is ligated to an oligonucleotide linker that is modified at one end with phosphoramidite and immobilized on the surface comprising Si0₂.