Patent Application
20120214162
The present invention relates generally to assay methods for the analysis of biopolymers. Mapping and sequencing of such biopolymers is contemplated herein.
A number of different approaches for sequencing nucleic acids exist. The traditional methods are the dideoxy-chain termination method described by Sanger et al., Proc Natl. Acad. Sci. USA, (1977) 74: 5463-67 and the chemical degradation method described by Maxam et al., Proc. Natl. Acad. Sci. USA, (1977) 74: 560-564. Of these two methods, the Sanger procedure has been the most widely used. The original Sanger method relied on radioactive labeling of the reaction products and separation of the reaction products by slab gel electrophoresis.
Both the Sanger and Maxam methods are time- and labor-intensive. The start of the Human Genome Project was the impetus for the development of improved, automated systems to perform Sanger sequencing. As a result, detection of fluorescence has replaced autoradiography and capillary electrophoresis has replaced the ultrathin slab gels originally used to separate reaction products. Automated sequencers have been developed and are capable of processing large numbers of samples without operator intervention.
The completion of the Human Genome Project has refocused the need for new technologies that are capable of rapidly and inexpensively determining the sequence of human and other genomes. There is has been much discussion in recent years about personalized medicine. The vision of personalized medicine involves each individual having his or her complete genome sequenced at high accuracy and using this information to guide clinical care, specifically for risk stratification of patients and pharmacogenomics.
In recent years, a number of technological advances have been developed enabling a great reduction in the cost of sequencing and substantially increasing the amount of sequence data produced. Most sequencing methods currently available utilize optical detection for the determination of the DNA sequence. The most prevalent sequencing methods are referred to as sequencing by synthesis (SBS).
Typical SBS methods involve the stepwise synthesis of a strand of DNA that is complementary to a template sequence from the target genome to be sequenced. The SBS methods can be divided into those that are performed in batch mode and those that are performed in real-time. The batch mode processes rely on the stepwise synthesis of the new DNA strand with the limitation that the synthesis is only allowed to proceed for one nucleotide position, for one nucleotide type, or for the combination of one nucleotide position and one nucleotide type. The incorporation of the nucleotide occurs in parallel for large numbers of templates. Detection is achieved using a variety of methods.
A batch mode SBS method utilizing a single nucleotide type is used by Roche for pyrosequencing with the 454 platform. The Roche technology (see, e.g., Margulies et al. (2005) Nature, 437:376-380; U.S. Pat. Nos. 6,274,320; 6,258,568; 6,210,891) utilizes pyrosequencing. The method depends on several enzymes and cofactors to produce luminescence when a nucleotide is incorporated. A single nucleotide species is introduced into a large number of small reaction vessels each containing multiple copies of a single template. The incorporation of the nucleotide is accompanied by light emission. When the reaction has run to completion, the reagents are washed from the reaction volumes and a next nucleotide and its required reagents are washed into the reactions. Each template is thus extended in an iterative fashion, one nucleotide at a time. Multiple incorporations of the same nucleotide require the quantitative determination of the amount of light emitted. Homopolymer tracts in templates may be difficult to accurately sequence as the incremental amount of light emitted for each subsequent position in the homopolymer becomes small compared to the total amount emitted.
In a second SBS method, platforms by Helicos (see, e.g., Quake et al Proc. Nat. Acad. Sci. USA (2003) 100: 3960-3964; U.S. Pat. Nos. 6,818,395; 6,911,345; 7,297,518; 7,462,449 and 7,501,245), Illumina (see, e.g., Bennett et al. Pharmacogenomics (2005) 6:373-382), and Intelligent Bio-Systems (see, e.g., Ju et al. Proc. Nat. Acad. Sci. USA (2006) 103:19635-19640) allow only the incorporation of a single nucleotide at each step. Template strands are attached to a solid support and a primer sequence is annealed. A polymerase is used to extend the primer to make a complement to the template. The nucleotides are derivatized such that after the incorporation of a single nucleotide, the growing strand is incapable of further extension. The nucleotides are further derivatized to make them fluorescent. In the Helicos technology, the four nucleotides are labeled with the same fluorescent tag. This requires that each nucleotide type be added separately. In contrast, the Illumina and Intelligent Bio-Systems technologies utilize four different fluorescent tags so that a mixture of all four derivatized nucleotides may be added at the same time. For both technologies, the incorporation of a nucleotide is accompanied by the appearance of fluorescence in the growing strand. In the case of Illumina, the wavelength of the fluorescence emission indicates the identity of the newly incorporated nucleotide. In the Helicos technology, only a single nucleotide type is added at each cycle. Thus, the appearance of fluorescence at a position on the solid support indicates the incorporation of the added nucleotide for that template. Templates that do not incorporate the nucleotide present in the reaction remain dark.
Following the observation of any incorporated fluorescence, the blocking groups and fluorescent tags are removed prior to the next cycle. Multiple cycles result in the acquisition of sequence data for many templates in a single run. The instrumentation typical for these technologies is said to allow for the automated acquisition of sequence information for hundreds of thousands to millions of templates in parallel.
SBS methods may also be performed in real-time. In real-time SBS, polymerase is used to incorporate fluorescently labeled nucleotides and the fluorescence is observed during DNA strand synthesis. The four nucleotides are labeled with different fluorescent tags. The fluorescent tags are attached to the terminal phosphate of the nucleotide triphosphate. During incorporation of the nucleotide into the growing strand the fluorophore is released to solution and the growing strand remains non-fluorescent. The identity of the incorporated strand is determined while the nucleotide resides in the active site of the enzyme and before the cleaved diphosphate is released to bulk solution.
The fluorescence of the incorporated nucleotide typically is measured in a background fluorescence from a much larger concentration of unincorporated nucleotide. Pacific Biosciences (see, e.g., U.S. Pat. Nos. 7,170,050; 7,302,146; 7,315,019; 7,476,503; and 7,476,504) identifies the incorporated nucleotide based on the residence time in the polymerase active site. Fluorescence emission from the active site for an appropriate time indicates incorporation and the emission wavelength determines the identity of the incorporated nucleotide. Polymerase is attached to the bottom of zero-mode waveguides. Zero-mode waveguides are reaction cells whose dimensions limit the passage of light from the excitation sources. Thus, only fluorescent tags close to the bottom surface of the reaction volume are excited. Visigen identifies the incorporated nucleotide through Fluorescent Resonant
Energy Transfer (FRET) between an acceptor in the polymerase active site and a fluorescent tag on the nucleotide (see, e.g., U.S. Pat. Nos. 7,211,414 and 7,329,492). Only nucleotides held in the active site of the polymerase show fluorescence. Incorporation is identified by the residence time of the fluorescence in the active site and the nucleotide identity is determined by the emission wavelength.
Other recently developed methods to sequence DNA rely on hybridization and ligation. Both the SOLiD and Complete Genomics technologies rely on the combination of hybridization and ligation. The SOLiD system (Life Technologies) immobilizes short template strands via an adapter. A primer and a pool of labeled oligonucleotides containing two fixed positions and six degenerate positions is hybridized to the template.
The primer hybridizes to the adaptor. Each pool consists of 16,384 different sequences. Four fluorescent dyes are used to label the oligonucleotides in a pool in a fashion that creates four subsets from the sixteen combinations at the two fixed positions. Thus, each fluorescent tag is associated with four of the sixteen possible combinations. Following hybridization, a ligase is added and any probes in the pool that hybridized contiguously with the primer are ligated to the primer. The fluorescence of the hybridized and ligated product is determined. The fluorescence defines which subset of sequences hybridized to the template and ligated to the primer. The terminal three bases and the associated fluorescent tag are cleaved from the hybridized and ligated oligonucleotide. Subsequent rounds of another round of hybridization, ligation, and cleavage are performed. In this first series of reactions, each cycle identifies a subset for the pair of nucleotides in the template that is 5 nucleotides downstream from subset of pairs that were identified in the last cycle. After several cycles, the primer, and the oligonucleotides that have been ligated to it, is washed off the template.
The entire procedure is repeated starting with a primer that is one nucleotide shorter than the original primer, then with primers that are two, three, and four nucleotides shorter than the original primer. These subsequent rounds shift the frame of interrogation so that the bases that make-up the template strand can be identified from the union between the two subsets of reaction that overlapped at that position.
Complete Genomics technology utilizes a similar hybridization and ligation method (see, e.g., US Patent Application Publication Nos. 20080234136; 20090005252; 20090011943; and 20090176652). In the Complete Genomics technology, a primer is hybridized to an adaptor that is attached to the end of the template. A series of pools of oligonucleotides is constructed. In each pool, the nucleotide at a single position is identified by using four-color fluorescence. The remaining positions are degenerate. The first pool is hybridized to the template. Oligonucleotides that hybridize adjacent to the primer are subsequently ligated. After washing excess oligonucleotides away, the fluorescence of the ligated oligonucleotide identifies the nucleotide at the defined position in that pool. The ligated primer and oligonucleotide are washed off the template and the process is repeated with the next pool of oligonucleotides that probe the next position down from the primer.
The SBS and hybridization-ligation methods generate short pieces or reads of DNA sequence. While the short reads can be used to re-sequence human genomes, they are not favorable for the de novo assembly of human genomes. With the recent realization that human genomes contain large numbers of inversions, translocations, duplications, and indels (e.g., mutations that include both insertions, deletions, and the combination thereof), the quality of human genome data from short reads is even more suspect. Genetic rearrangements are even more prevalent in cancer.
While variations of the short read technologies that incorporate paired-end reads have been proposed and the length of the sequence data from these technologies has increased incrementally over the last two years, it is clear that longer read technologies are necessary for the accurate assembly of human genome data.
In addition to the undesirable nature of short reads, all of the DNA sequencing methods described above employ optical detection. The throughput of optical methods limits the ultimate performance characteristics of any of these sequencing technologies. Optical methods are capable of identifying single molecules. However, the time required to observe and accurately identify events is typically too slow to meet the need for higher throughput. While the current generation of sequencing technologies has lowered the cost of sequencing by orders of magnitude in comparison to the methods used to sequence the first human genomes, the methods remain too slow, costly, and inaccurate for routine analysis of human genomes.
A need therefore exists for efficient methods and devices capable of rapid and accurate nucleic acid sequencing for de novo assembly of human genomes. It is desirable to have long read lengths and to use as little nucleic acid template as possible. Moreover, single-molecule optical detection of DNA has limitations with respect to sensitivity and speed.
Thus, there remains a need for improved methods and devices for the analysis of biopolymers, including methods and devices for mapping and sequencing such biopolymers.
Embodiments of the invention provide assay methods for preparing analyte samples for mapping and sequencing using nanopore, micro-channel or nano-channel analysis devices.
In an aspect, an embodiment of the invention includes a method for preparing a biomolecule analyte. The method includes: a) providing a single-stranded DNA or RNA template; b) hybridizing a first plurality of identical, sequence specific oligonucleotide probes to the template, each probe having a 5′ end and a 3′ end, to thereby form an analyte having at least one single-stranded region and at least two duplex regions; c) conducting a base extension reaction in the at least one single-stranded region from the 3′ end of a hybridized probe toward the 5′ end of an adjacent hybridized probe; d) terminating the base-extension reaction such that there remains for each single-stranded region a single-stranded portion thereof adjacent to the 5′ end of each hybridized probe, and e) reacting the resulting analyte with a binding moiety that selectively binds to the at least one single-stranded portion to thereby prepare the biomolecule analyte.
One or more of the following features may be included. The probes may include single-stranded DNA and/or RNA. The base extension reaction may be performed by a DNA or RNA polymerase. The binding moiety may include a protein, such as RecA, T4 gene 32 protein, f1 geneV protein, human replication protein A, P13 single-stranded binding protein, adenovirus DNA binding protein, and/or E. coli single-stranded binding protein. A length of each probe may be selected from a range of 4 to 12 bases.
Steps a-e may be performed sequentially. Steps a-e may be repeated sequentially by replacing the first plurality of probes with a subsequent plurality of different unique probes. At least a portion of the probes in the first plurality of probes has attached thereto a detectable tag.
The biomolecule analyte may be configured for detection of positional information in a nanopore system. Changes in an electrical property across a nanopore may be monitored as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety. Regions of the biomolecule analyte including or lacking the binding moiety may be differentiated based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.
The biomolecule analyte may be configured for detection of positional information in a fluidic channel system. The fluidic channel system may include a micro-channel or a nano-channel. Changes in an electrical property across a fluidic channel may be monitored as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety. Regions of the biomolecule analyte including or lacking the binding moiety may be differentiated based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.
A sequence of at least a portion of the single-stranded DNA or RNA template may be determined according to the determined binding moiety locations.
Sequence-specific oligonucleotide analog probes such as LNAs, PNAs or 2′-methoxy nucleotide analogs may be substituted for the sequence specific oligonucleotide probes. The first plurality of identical, sequence specific oligonucleotide probes may be replaced by a pool of sequence specific oligonucleotide probes including at least a first plurality of identical, sequence specific oligonucleotide probes and a second plurality of identical, sequence specific oligonucleotide probes, with the probes of the second plurality being different from the probes of the first plurality.
In another aspect, embodiments of the invention includes a method for preparing a biomolecule analyte by: a) providing a single-stranded DNA template; b) hybridizing a first plurality of identical, sequence specific RNA probes to the template, each probe having a 5′ end and a 3′ end, to thereby form an analyte having at least one single-stranded region and at least one duplex region; c) conducting a base extension reaction in the at least one single-stranded region from the 3′ end of a hybridized probe; d) allowing the base-extension reaction to fill each single-stranded region on the analyte; e) removing the RNA probes to provide the analyte with at least one single-stranded segment in the region to which an RNA probe had been hybridized; and f) reacting the resulting analyte with a binding moiety that selectively binds to the single-stranded segment, to thereby prepare the biomolecule analyte.
One or more of the following features may be included. The base extension reaction may be performed by a DNA or RNA polymerase. The binding moiety may include a protein, such as RecA, T4 gene 32 protein, f1 geneV protein, human replication protein A, P13 single-stranded binding protein, adenovirus DNA binding protein, and/or E. coli single-stranded binding protein. A length of each probe may be selected from a range of 4 to 12 bases.
Steps a-f may be performed sequentially. Steps a-f may be repeated sequentially by replacing the first plurality of probes with a subsequent plurality of different unique probes.
The RNA probes may be removed by reacting the analyte with hydroxyl ions.
The biomolecule analyte may be configured for detection of positional information in a nanopore system. Changes in an electrical property across a nanopore may be monitored as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety. Regions of the biomolecule analyte including or lacking the binding moiety may be differentiated based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.
The biomolecule analyte may be configured for detection of positional information in a fluidic channel system. The fluidic channel system may include a micro-channel or a nano-channel. Changes in an electrical property across a fluidic channel may be monitored as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety. Regions of the biomolecule analyte including or lacking the binding moiety may be differentiated based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.
A sequence of at least a portion of the single-stranded DNA or RNA template may be determined according to the determined binding moiety locations.
The first plurality of identical, sequence specific RNA probes may be replaced by a pool of sequence specific RNA probes including at least a first plurality of identical, sequence specific RNA probes and a second plurality of identical, sequence specific RNA probes, the probes of the second plurality being different from the probes of the first plurality.
In yet another aspect, embodiments of the invention include a method for preparing a biomolecule analyte. The method includes: a) providing a single-stranded DNA or RNA template; b) hybridizing a first plurality of identical, sequence specific oligonucleotide probes to the template, to thereby form an analyte having at least one single-stranded region and at least one duplex region; and c) reacting the resulting analyte with a binding moiety that selectively binds to the at least one duplex region to thereby prepare the biomolecule analyte.
One or more of the following features may be included. The probes may include single-stranded DNA and/or RNA. The binding moiety may include a protein, such as RecA, T4 gene 32 protein, f1 geneV protein, human replication protein A, Pf3 single-stranded binding protein, adenovirus DNA binding protein, and/or E. coli single-stranded binding protein. A length of each probe may be selected from a range of 4 to 12 bases.
Steps a-c may be performed sequentially. Steps a-c may be repeated sequentially by replacing the first plurality of probes with a subsequent plurality of different unique probes. At least a portion of the probes in the first plurality of probes has attached thereto a detectable tag.
The biomolecule analyte may be configured for detection of positional information in a nanopore system. Changes in an electrical property across a nanopore may be monitored as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety. Regions of the biomolecule analyte including or lacking the binding moiety may be differentiated based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.
The biomolecule analyte may be configured for detection of positional information in a fluidic channel system. The fluidic channel system may include a micro-channel or a nano-channel. Changes in an electrical property across a fluidic channel may be monitored as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety. Regions of the biomolecule analyte including or lacking the binding moiety may be differentiated based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.
A sequence of at least a portion of the single-stranded DNA or RNA template may be determined according to the determined binding moiety locations.
Sequence specific oligonucleotide analog probes, such as LNAs, PNAs and 2′-methoxy nucleotide analogs, may be substituted for the sequence specific oligonucleotide probes. The first plurality of identical, sequence specific oligonucleotide probes may be replaced by a pool of sequence specific oligonucleotide probes including at least a first plurality of identical, sequence specific oligonucleotide probes and a second plurality of identical, sequence specific oligonucleotide probes, the probes of the second plurality being different from the probes of the first plurality.
In another aspect, embodiments of the invention include a method for preparing a biomolecule analyte. The method includes: a) providing a single-stranded DNA or RNA template; b) hybridizing a first plurality of identical, sequence specific oligonucleotide probes to the template, to thereby form an analyte having at least one single-stranded region and at least one duplex region; and c) reacting the resulting analyte with a binding moiety that selectively binds to the at least one single-stranded region, to thereby prepare the biomolecule analyte.
One or more of the following features may be included. The probes may include single-stranded DNA and/or RNA. The binding moiety may include a protein, such as RecA, T4 gene 32 protein, f1 geneV protein, human replication protein A, Pf3 single-stranded binding protein, adenovirus DNA binding protein, and/or E. coli single-stranded binding protein. A length of each probe may be selected from a range of 4 to 12 bases.
Steps a-c may be performed sequentially. Steps a-c may be repeated sequentially by replacing the first plurality of probes with a subsequent plurality of different unique probes. At least a portion of the probes in the first plurality of probes has attached thereto a detectable tag.
The biomolecule analyte may be configured for detection of positional information in a nanopore system. Changes in an electrical property across a nanopore may be monitored as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety. Regions of the biomolecule analyte including or lacking the binding moiety may be differentiated based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.
The biomolecule analyte may be configured for detection of positional information in a fluidic channel system. The fluidic channel system may include a micro-channel or a nano-channel. Changes in an electrical property across a fluidic channel may be monitored as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety. Regions of the biomolecule analyte including or lacking the binding moiety may be differentiated based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.
A sequence of at least a portion of the single-stranded DNA or RNA template may be determined according to the determined binding moiety locations.
Sequence specific oligonucleotide analog probes such as LNAs, PNAs and 2′-methoxy nucleotide analogs may be substituted for the sequence specific oligonucleotide probes. The first plurality of identical, sequence specific oligonucleotide probes may be replaced by a pool of sequence specific oligonucleotide probes including at least a first plurality of identical, sequence specific oligonucleotide probes and a second plurality of identical, sequence specific oligonucleotide probes, the probes of the second plurality being different from the probes of the first plurality.
In some embodiments, the probes are single-stranded DNA or RNA, and they may optionally be provided with tags that enhance detection in analysis devices. Based on the nature of the tags, the binding moiety may bind to the tags as well as to regions of the analyte, further enhancing detection. Alternatively, the probes may include oligonucleotide analogs such as Locked Nucleic Acids (LNAs), Peptide Nucleic Acids (PNA s) or 2′-methoxy nucleotide analogs. Base extension reactions are performed using DNA polymerase or RNA polymerase, as appropriate.
In any of the above-described embodiments, one or more of the features described in this paragraph and in the paragraphs that follow may be included. The process steps may be repeated using subsequent pluralities of matching probes having a known binding selectivity different from the known binding selectivity of the first plurality of probes. The first plurality of probes may be hybridizing oligonucleotides having n number of bases, with n preferably ranging from 4 to 12. Each process may be repeated sequentially by replacing the first plurality of probes with a subsequent plurality of each of the different unique probes within the entire library of 4n n-mer probes. A portion of the library of 4n n-mer probes may be used, such as about 85%, 75%, 65%, 55%, 45%, or 33% of the library. The sequential repetition of the process may be conducted in a linear or parallel series of reactions.
Alternatively, rather than employing a single plurality of identical, sequence specific oligonucleotide probes, a pool of probes comprising a first plurality of identical, sequence specific oligonucleotide probes and a second plurality of identical, sequence specific oligonucleotide probes, different from the first plurality, may be employed.
The biomolecule to be analyzed may be DNA or RNA. The binding moiety may be a protein. Examples of suitable proteins include RecA, T4 gene 32 protein, f1 geneV protein, human replication protein A, Pf3 single-stranded binding protein, adenovirus DNA binding protein, and E. coli single-stranded binding protein.
The products of the assay methods described herein may be analyzed in an apparatus using a nanopore or a fluidic channel such as a micro-channel or nano-channel to detect probes or probe positions on the biomolecule being analyzed. Suitable apparatus are described, for example, in U.S. patent application Ser. No. 12/789,817, published as U.S. Publication No. 2010/0310421, incorporated herein by reference in its entirety Such systems use electrical detection methods to determine the presence and/or the relative position of the oligonucleotide probes. The products of the assay methods described herein may be sequenced in accordance with sequencing methods such as those described in U.S. patent application Ser. No. 13/292,415, incorporated herein by reference in its entirety.
An electrical property across a nanopore or fluidic channel as the analyte translocates therethrough may be monitored, with changes in the electrical property being indicative of regions on the analyte including or lacking the binding moiety. Furthermore, differentiating between regions on the analyte including or lacking the binding moiety, allows a determination of binding moiety locations on the analyte.
a)-5(d) are a schematic depiction of an assay method in accordance with an embodiment of the invention in which single-stranded DNA (“ssDNA”) probes are bound to a single-stranded DNA or RNA analyte, a base extension reaction is carried out, and a binding moiety which binds to single-stranded portions of the resulting analyte is employed.
a)-6(d) are a schematic depiction of an assay method in accordance with an embodiment of the invention in which tagged ssDNA probes are bound to a single-stranded DNA or RNA analyte, a base extension reaction is carried out, and a binding moiety which binds to single-stranded portions of the resulting analyte is employed.
a)-7(f) are a schematic depiction of an assay method in accordance with an embodiment of the invention in which RNA probes are bound to a single-stranded DNA analyte, a base extension reaction is carried out, the RNA probes are removed, and a binding moiety which binds to single-stranded portions of the resulting analyte is employed.
a)-8(c) are a schematic depiction of an assay method in accordance with an embodiment of the invention in which oligonucleotide probes are bound to a single-stranded DNA or RNA analyte, and a binding moiety is bound to duplex structures formed by the probes.
a)-9(c) are a schematic depiction of an assay method in accordance with an embodiment of the invention in which tagged oligonucleotide probes are bound to a single-stranded DNA or RNA analyte, and a binding moiety is bound to duplex structures formed by the probes.
a)-10(c) are a schematic depiction of an assay method in accordance with an embodiment of the invention in which oligonucleotide probes are bound to a single-stranded DNA or RNA analyte, and a binding moiety is bound to the analyte in gaps between the probes.
a) is a schematic depiction of an assay method in accordance with an embodiment of the invention showing a DNA molecule having a binding moiety region in a nanopore apparatus.
b) is a schematic depiction of an assay method in accordance with an embodiment of the invention showing a current measurement waveform as a DNA molecule having a binding moiety region translocates through the nanopore apparatus of
a) is a schematic depiction of an assay method in accordance with an embodiment of the invention showing an electrical potential measurement as a DNA molecule having a binding moiety region enters a detection volume in the apparatus of
b) is a schematic depiction of an assay method in accordance with an embodiment of the invention showing an electrical potential measurement as a binding moiety region on a DNA molecule enters a detection volume in the apparatus of
c) is a schematic depiction of an assay method in accordance with an embodiment of the invention showing an electrical potential measurement as a binding moiety region on a DNA molecule exits a detection volume in the apparatus of
d) is a schematic depiction of an assay method in accordance with an embodiment of the invention showing an electrical potential measurement as a DNA molecule having a binding moiety region exits a detection volume in the apparatus of
As used in this description and the accompanying claims, the following terms shall have the meanings given, unless the context indicates otherwise:
A “biomolecule analyte” or “target analyte” means a biomolecule, for example, having sequence information that is to be determined using embodiments of the present invention. The target may be a biomolecule such as, for example, deoxyribonucleic acid, a ribonucleic acid, a protein, or a polypeptide. The target may be single-stranded or double-stranded.
A “probe” means any molecule or assembly of molecules capable of sequence-specific covalent or non-covalent binding to an analyte.
A “tag” means a moiety that is attached to a probe in order to make the probe more visible to a detector. These tags may be, for example, proteins, double-stranded DNA, single-stranded DNA or other molecules. Exemplary tags that will bind RecA include single-stranded or double-stranded DNA. Another useful tag may be a dendrimer, for example. Optionally, tags have either a larger volume than the probe or a different charge so that they slow translocation of the biomolecule through a nanopore, or through a nano-channel or micro-channel.
Disclosed herein are methods that increase the signal-to-noise ratio in translocation detection of biomolecules that have been hybridized to probes. In one embodiment, a single-stranded biomolecule may be hybridized with a probe. The hybridized biomolecule may then be incubated with a protein or enzyme that binds to the biomolecule and forms at least a partial coating along the biomolecule.
Coated biomolecules typically have greater uniformity in their translocation rates, which leads to a decrease in positional error and thus more accurate sequencing. Due to its increased diameter, a coated biomolecule generally translocates through a sequencing system at a slower speed than a non-coated biomolecule. The translocation is preferably slow enough so that a signal can be detected during its passage from a first chamber into a second chamber. The translocation rate or frequency may be regulated by introducing a salt gradient between the chambers. Exemplary salt concentration ratios of the cis to the trans side of the chamber may include, but are not limited to, 1:2, 1:4, 1:6, and 1:8. For example, salt concentrations may range from about 0.5 M KCl to about 1 M KCl on the cis side and from about 1 M KCl to about 4 M KCl on the trans side. The signal is preferably strong enough to be detected using known methods or methods described herein. Exemplary signal-to-noise ratios include, but are not limited to, 2:1, 5:1, 10:1, 15:1, 20:1, 50:1, 100:1, and 200:1. With a higher signal-to-noise ratio, a lower voltage may be used to effect translocation.
In one embodiment, a biomolecule of interest is hybridized with the entire library of probes of a given length. For example, the biomolecule of interest can be hybridized with the entire universe of 4096 (i.e., 46) possible six-mers. The hybridization can be done sequentially (i.e., one probe after another) or in parallel (i.e., a plurality of biomolecules of interest are each separately hybridized simultaneously with each of the possible probes.) Alternatively, the probes can be separated from each other in both space and time. Additionally, more than one probe type may be hybridized to the same biomolecule of interest at the same time.
The set of probes used to perform the sequencing may be a subset of the complete library of probes of a given length, such as about 85%, 75%, 65%, 55% , 45%, or 33% of the library. For instance, if sequencing is performed on a biomolecule that starts as double-stranded DNA, then only one-half of the probes that make up a library may be needed. Other subsets of the library may be designed to allow sequencing as well. If some information concerning the target sequence is known prior to performing the sequencing reaction, it may be possible to use a small subset of the total library. For instance, if the sequencing reaction is being performed to determine if single nucleotide polymorphisms are present with respect to a reference sequence, then a small number of probes with respect to the complete library may be used. Alternatively, the set of probes may not all be the same length. In an embodiment, a set of at least two probes may be used for hybridization, rather than an entire library of probes or subset thereof. In another embodiment, probes may be separated by (GC) content or other determinants of probe binding strength, in order to allow for optimization of reaction conditions. By separating the probes based on relative properties, multiple probes may be incorporated into a single hybridization reaction. Further, the probes may be grouped based on their related optimum reaction environment preferences. In yet another embodiment, pools of probes may be simultaneously hybridized to a biomolecule of interest. A pool of probes is a group of probes of different composition, each of which may likely be present in many copies. The composition of the probes may be chosen so as to reduce the chance of competitive binding to the biomolecule of interest. Alternatively, the composition of multiple pools may be chosen so that the same competitive binding is not present in all pools occupied by a single probe.
In still another embodiment, the probes may be attached to tags, making electrical fluctuations more noticeable as the hybridized probes translocate through the sequencing system. In addition, different tags may be used to help distinguish among the different probes. These tags may be proteins, double-stranded DNA, single-stranded DNA or other molecules.
It should be understood that the invention is not intended to be limited strictly to DNA and RNA oligonucleotide probes. Rather, it is envisioned that oligonucleotide analog probes such as those comprising LNAs, PNAs and 2′-methoxy nucleotide analogs may be used as well.
In embodiments of the present invention, the biomolecule may be hybridized with sequence-specific probes prior to being reacted with a binding moiety such as a protein. The probes may or may not have tags attached to them. If the probe has an attached tag composed of single- or double-stranded DNA, the binding moiety, such as the protein RecA, may coat (i) the single-stranded target, (ii) the double-stranded regions where hybridization between the single-stranded target and (iii) the probe has occurred, and the tag attached to the probe. Alternatively, the bound probes and associated tags may have a different affinity for the binding moiety than for the biomolecule. If the tags have an essentially equal affinity for the binding moiety, then both the tag and the target may be coated. If the tag or probe has a greater affinity for the binding moiety, selective coating may be achieved. If the tag or probe has a lower affinity for the binding moiety, it may selectively coat regions of the biomolecule that do not have probe bound. Since any region with bound protein will have a larger signal, differentiation of the hybridized and non-hybridized regions allows for greater accuracy in determining the position of hybridization.
The translocation of biomolecule/protein complexes through a nanopore, a nano-channel or a micro-channel sequencing system may include detecting an electrical signal indicative of the passage of coated regions. In one embodiment, the signal detected may be formed by passage of the tagged region of the biomolecule through the sequencing system. The time for translocation may be indicative of the length of the biomolecule. The detection step may discriminate between coated, uncoated, or multiply coated regions, as a coated region may have a signal about ten times that of an uncoated region. Increased signal-to-noise may increase confidence for the detection of the probes. Positional information of probe binding to target biomolecule allows for the mapping or sequencing of the biomolecule analyte.
In one embodiment, the process of sequencing a biomolecule such as single-strands of DNA or RNA using one or more probes may performed as follows. Suitable processes are also described in U.S. Ser. No. 11/538,189, published as U.S. Publication No. 2007/0190542, incorporated herein by reference in its entirety. Referring to
Traditionally, in determining the particular arrangement of the bases 6 and thereby the sequences of the molecules, a process called hybridization may be utilized. The hybridization process is the coming together, or binding, of two genetic sequences with one another. This process is predictable because the bases 6 in the molecules do not share an equal affinity for one another. T (or U) bases favor binding with A bases while C bases favor binding with G bases. Binding is mediated via hydrogen bonds that exist between the opposing base pairs. For example, A binds to T (or U) using two hydrogen bonds, while C binds to G using three hydrogen bonds.
A hybridizing oligonucleotide, i.e., a probe, may be used to determine and identify the sequence of bases in the molecule of interest.
In this regard, the total number of unique probes 10 in a library is dependent upon the number of bases 12 contained within each probe 10 and the number of different types of bases in the probes. If only the four natural bases are used in probe 10, the total number of probes in the library is determined by the formula 4″ (four raised to the n power) where n is equal to the total number of bases 12 in each probe 10. Formulas for other arrangements or types of bases are well known in the art. Accordingly, the size of the probe library can be expressed as 4″ n-mer probes 10. For the purpose of illustration, in the context of a six-mer probe, the total number of possible unique, identifiable probe combinations includes 46 (four raised to the sixth power) or 4096 unique six-mer probes 10. The inclusion of non-natural bases allows for the creation of probes that have spaces or wildcards therein in a manner that expands the versatility of the library, while reducing the number of probes that may be needed to reach the final sequence result. Probes that include universal bases organized into patterns with natural bases may also be used, for example those described in U.S. Pat. Nos. 7,071,324, 7,034,143, and 6,689,563, incorporated herein by reference in their entireties.
The process of hybridization using probes 10, as depicted in
Embodiments of the present invention relate to improved methods for the preparation of biomolecule strands for analysis. In one embodiment, shown in
The biomolecule analyte 15 is shown in
Following the hybridizing step, a base extension reaction, such as a primer extension reaction, utilizing for example, a polymerase and one or more nucleotides, is performed as depicted in
Finally, as depicted in
The protein RecA from E. coli typically binds single- or double-stranded DNA in a cooperative fashion to form filaments containing the DNA in a core and an external sheath of protein (McEntee, K.; Weinstock, G. M.; Lehman, I. R. Binding of the RecA Protein of Escherichia coli to Single- and Double-Stranded DNA. J. Biol. Chem. 1981, 256, 8835, incorporated herein by reference in its entirety). DNA has a diameter of about 2 nm, while DNA coated with RecA has a diameter of about 10 nm. The persistence length of the DNA increases to around 950 nm, in contrast to 0.75 nm for single-stranded DNA or 50 nm for double-stranded DNA. T4 gene 32 protein is known to cooperatively bind single-stranded DNA (Alberts, B. M.; Frey, L. T4 Bacteriophage Gene32: A Structural Protein in the Replication and Recombination of DNA. Nature, 1970, 227, 1313-1318, incorporated herein by reference in its entirety). E. coli single-stranded binding protein binds single-stranded DNA in several forms depending on salt and magnesium concentrations (Lohman, T. M.; Ferrari, M. E. Escherichia Coli Single-Stranded DNA-Binding Protein: Multiple DNA-Binding Modes and Cooperativities. Ann. Rev. Biochem. 1994, 63, 527-570, incorporated herein by reference in its entirety). The E. coli single-stranded binding protein may form a varied coating on the biomolecule. The f1 geneV protein is known to coat single-stranded DNA (Terwilliger, T.C. Gene V Protein Dimerization and Cooperativity of Binding of poly(dA). Biochemistry 1996, 35, 16652, incorporated herein by reference in its entirety), as is human replication protein A (Kim, C.; Snyder, R. O.; Wold, M. S. Binding properties of replication protein A from human and yeast cells. Mol. Cell Biol. 1992, 12, 3050, incorporated herein by reference in its entirety), Pf3 single-stranded binding protein (Powell, M. D.; Gray, D. M. Characterization of the Pf3 single-strand DNA binding protein by circular dichroism spectroscopy. Biochemistry 1993, 32, 12538, incorporated herein by reference in its entirety), and adenovirus DNA binding protein (Tucker, P. A.; Tsernoglou, D.; Tucker, A. D.; Coenjaerts, F. E. J.; Leenders, H.; Vliet, P. C. Crystal structure of the adenovirus DNA binding protein reveals a hook-on model for cooperative DNA binding. EMBO J. 1994, 13, 2994, incorporated herein by reference in its entirety). The protein-coated DNA may then be translocated through a nanopore as has been demonstrated with RecA bound to double-stranded DNA (Smeets, R. M. M.; Kowalczyk, S. W.; Hall, A. R.; Dekker, N. H.; Dekker, C. Translocation of RecA-Coated Double-Stranded DNA through Solid-State Nanopores. Nano Lett. 2009, incorporated herein by reference). Translocation of protein bound to single-stranded DNA is contemplated. The protein coating functions in the same manner for single-stranded DNA and double-stranded DNA.
It is important that the binding moiety 50 be distinguishable from duplex regions when the analyte is introduced to a nanopore or micro- or nanochannel sequencing system. As such, the locations of the binding moiety 50 on each fragment may be identified, leading to the location of each probe, since the binding moiety will end adjacent to the 5′ end of each probe. Since the positions of the probes may then be readily determined, and as the analyte portions containing the probes will have regions complementary to the known probe sequences, one may determine the sequence and location of specific domains on the analyte. Numerous maps may be created, corresponding to the positions of different probes. The resulting maps may be combined and utilized to determine broader sequence information for the analyte.
A similar embodiment is depicted in
The biomolecule fragment 20 to be analyzed and probes 70 are depicted prior to hybridization in
The analyte 60 is shown in
Following the hybridization step, a base extension reaction is performed as depicted in
Finally, as depicted in
Another embodiment is depicted in
The analyte 25 is shown in
Following the hybridizing step, a DNA base extension reaction is performed as depicted in
The analyte 25 of
Finally, as depicted in
The analyte 100 is shown in
Next, unlike the embodiments of
Similarly, in
The analyte 120 is shown in
As is shown in
Another embodiment, depicted in
The analyte 130 is shown in
In
Mapping of target analytes prepared using the methods of embodiments of the present invention may be carried out using electrical detection methods employing nanopores, nano-channels or micro-channels using the methods described in U.S. patent application Ser. No. 12/789,817.
In one embodiment, current is measured during translocation of a DNA strand through a nanopore as shown in
Specifically, for nanopore 150, a measurable current produced by electrodes 152, 154 runs parallel 160 to the movement of the target analyte 25. In this example, the target analyte 25, i.e., biomolecule analyte, is made using the method depicted in
In
In another embodiment, an electrical property such as electrical potential or current is measured during translocation of a DNA strand through a nano-channel or micro-channel as shown in
A first pair of electrodes 304, 304′ is connected to a current source 306 and positioned in a spaced apart relationship in the channel. These electrodes provide an electrical current along the channel and may be used to provide or enhance a driving force 308 to a target analyte 25, i.e., biomolecule analyte, in the channel. Other driving forces such as pressure or chemical gradients are contemplated as well. A second pair of electrodes 312, 312′, i.e., detector electrodes, is positioned preferably substantially perpendicular to the channel in a spaced apart relationship to define a detection volume 314. The second pair of electrodes 312, 312′, is connected to a detector 316, such as a voltmeter, which monitors an electrical property in the detection volume 314. In the embodiment where the detector 316 is a voltmeter, a drop in electrical potential, produced by the first pair of electrodes 304, 304′, is measured across the detection volume 314. As in the case of the nanopore apparatus, in this example, a target analyte 25 is made using the method depicted in
The operation of the device is depicted schematically in
Prior to the entry of the analyte 25 into the detection volume 314, a substantially constant voltage 322 is measured across the detection volume. This voltage is shown in the waveform 320 of
b) shows the device and waveform 320 once the portion of the analyte 25 including the binding moiety has entered the detection volume 314. Due to its increased volume, entry of the binding moiety into the detection volume 314 causes a further interruption or decrease in the electrical property measured in the detection volume. This further interruption or decrease causes a second trough 326 to be exhibited in the waveform 320.
In
Finally, as shown in
Another embodiment of a fluidic channel apparatus is shown in
A first pair of electrodes 404, 404′ is connected to a current source 406 and positioned in a spaced apart relationship in the channel. These electrodes provide an electrical current along the channel and may be used to provide or enhance a driving force 408 to an analyte 25 in the channel. Other driving forces such as pressure or chemical gradients are contemplated as well. Multiple detector electrodes 412, 414, 416, 418, are positioned preferably perpendicular to the channel in a spaced apart relationship to define a plurality of detection volumes between adjacent detector electrodes. Thus, as seen in
It should be understood that number of detector electrodes and detection volumes is not intended to be limited to those depicted in
Both the nanopore apparatus and the fluidic channel apparatus allow detection of an analyte as well as detection of a binding moiety present on the analyte. Furthermore, relative or absolute positional information of the binding moiety may be obtained. Since, in each of the embodiments depicted herein, the relative or absolute position of the binding moiety is, at the very least, suggestive of the relative or absolute position of a known probe, embodiments of the invention allow determination of the location of the known recognition sequence of the probe. This in turn, allows the biomolecule to be mapped. The repeated use of different probes allows greater complexity, i.e., multiple recognition sequences, to be combined. Multiple maps and sequence information produced using multiple probes may be generated.
Those skilled in the art will readily appreciate that all parameters listed herein are meant to be exemplary and actual parameters depend upon the specific application for which the methods and materials of embodiments of the present invention are used. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described.
The described embodiments of the invention are intended to be merely exemplary and numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in the appended claims.
This application claims priority to and the benefit of U.S. Provisional Patent Application No. 61/441,945, filed Feb. 11, 2011, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61441945 | Feb 2011 | US |
