US20060252077A1 - Stabilizing a nucleic acid for nucleic acid sequencing - Google Patents
Stabilizing a nucleic acid for nucleic acid sequencing Download PDFInfo
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- US20060252077A1 US20060252077A1 US11/413,381 US41338106A US2006252077A1 US 20060252077 A1 US20060252077 A1 US 20060252077A1 US 41338106 A US41338106 A US 41338106A US 2006252077 A1 US2006252077 A1 US 2006252077A1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Definitions
- the invention provides methods for sequencing a nucleic acid comprising stabilizing a primer/target nucleic acid duplex attached to a substrate.
- methods of the invention comprise the use of a dual-anchored primer/target nucleic acid duplex or stabilizing molecule.
- Cancer is a disease that is rooted in heterogeneous genomic instability. Most cancers develop from a series of genomic changes, some subtle and some significant, that occur in a small subpopulation of cells. Knowledge of the sequence variations that lead to cancer will lead to an understanding of the etiology of the disease, as well as ways to treat and prevent it.
- An essential first step in understanding genomic duplexity is the ability to perform high-resolution sequencing.
- nucleic acid sequencing Various approaches to nucleic acid sequencing exist.
- One conventional way to do bulk sequencing is by chain termination and gel separation, essentially as described by Sanger et al., Proc. Natl. Acad. Sci., 74(12): 5463-67 (1977). That method relies on the generation of a mixed population of nucleic acid fragments representing terminations at each base in a sequence. The fragments are then run on an electrophoretic gel and the sequence is revealed by the order of fragments in the gel.
- Another conventional bulk sequencing method relies on chemical degradation of nucleic acid fragments. See, Maxam et al., Proc. Natl. Acad. Sci., 74: 560-564 (1977).
- methods have been developed based upon sequencing by hybridization. See, e.g., Drrnanac, et al., Nature Biotech., 16: 54-58 (1998).
- the invention generally provides methods and surfaces for nucleic acid sequencing comprising stabilized primer/target nucleic acid duplexes on a surface.
- Methods of the invention generally contemplate the use of a primer/target nucleic acid duplex in which each of the primer and the template contain a molecule having a binding partner on the substrate.
- the primer/target is stabilized on the surface by binding of both the primer and the template to the surface.
- Binding pairs for use in the invention are any molecular pair that can be bound to a surface and attached to a nucleic acid.
- Some examples of preferred pairs include ligand/receptor, affinity pairs, antigen/antibody, and carbohydrate/lectin.
- biotin/streptavidin, digoxigenin/anti-digoxigenin, and dinitrophenol/anti-dinitrophenol perform well in the invention.
- Other pairs are apparent to the skilled artisan based upon the description of the invention provided below.
- the primer contains a member of a binding pair at its 5′ terminus
- the template contains a member of a binding pair at its 3′ terminus
- the primer contains a member of a binding pair at its 3′ terminus
- the template contains a member of a binding pair at its 5′ terminus.
- the template and primer may contain the same type or species of binding pair or they may contain separate types or species. Binding may occur to a single species of binding partner or to separate members of the same species.
- both the template and the primer are bioinylated at opposite ends oriented to the surface (i.e., one at the 3′ end and one at the 5′ end) and the two biotin molecules adhere to the same streptavidin molecule (which has capacity to bind four biotins) on the surface.
- the two biotins adhere to separate streptavidin molecules spaced closely together on the surface.
- the primer is attached to a member of a first binding pair and the template is attached to a member of a second binding pair.
- the first member attaches to its mate on the surface
- the second member attaches to its separate mate on the surface.
- the combination of two separate mating pairs reduces loss of the hybrid due to either the template or the primer dissociating. It is apparent to the skilled artisan based upon this disclosure that any combination of binding pairs works to stabilize hybrid binding to a surface.
- template and primer may have attached separate species of binder that, although distinct, bind to the same surface-bound mate.
- the invention comprises methods for sequencing nucleic acids using stabilized, support-bound primer/template hybrids as described above.
- methods of the invention comprise template-dependent sequencing by synthesis using a polymerase capable of adding nucleotides to the primer in a template-dependent fashion.
- the invention is particularly useful for single molecule nucleic acid sequencing in which primer/template duplex is attached to a substrate such that the duplex is individually optically resolvable. Individual strand sequence is determined by detecting ordered template-dependent nucleotide incorporation into the primer and compiling a sequence of the template based upon the order of incorporated nucleotides.
- the invention also provides for the use of a stabilizing molecule in template-dependent sequencing.
- Stabilizing molecules useful in the invention include, for example, locked nucleic acid (“LNA”) analogs and peptide nucleic acid (“PNA”) analogs.
- LNA locked nucleic acid
- PNA peptide nucleic acid
- a stabilizing molecule increases the affinity and specificity of the primer/target nucleic acid bond, and increases the melting temperature of the primer/target nucleic acid duplex or the specificity of incorporation of a nucleotide into the primer in a sequencing by synthesis reaction.
- An example of a locked nucleic acid is shown in FIG. 4 . Both locked nucleic acid and peptide nucleic acid analogs increase the melting temperature of the primer/template duplex and, therefore, confer stability on the hybrid, whether or not the anchoring strategies described above are used.
- Polymerases useful in the invention include any polymerizing agent capable of catalyzing a template-dependent addition of a nucleotide or nucleotide analog to a primer.
- a DNA polymerase an RNA polymerase, or a reverse transcriptase can be used.
- a thermophilic polymerase is used, such as ThermoSequenase®, 9° NTM, Taq, Tfl, Tth, Tli, Therminator, or Pfu.
- the invention provides for the primer/target nucleic acid duplex to be exposed to the polymerase and nucleotide at a temperature between about 30° and about 80° Celsius.
- a preferred polymerase is a Klenow fragment having reduced 3′-5′ exonuclease activity.
- Nucleotides useful in the invention include any nucleotide or nucleotide analog, whether naturally-occurring or synthetic.
- preferred nucleotides are adenine, cytosine, guanine, uracil, or thymine bases; xanthine or hypoxanthine, 5-bromouracil, 2-aminopurine, deoxyinosine, or methylated cytosine, such as 5-methylcytosine, and N4-methoxydeoxycytosine.
- bases of polynucleotide mimetics such as methylated nucleic acids, e.g., 2′-O-methRNA, peptide nucleic acids, modified peptide nucleic acids, locked nucleic acids and any other structural moiety that can act substantially like a nucleotide or base, for example, by exhibiting base-complementarity with one or more bases that occur in DNA or RNA and/or being capable of base-complementary incorporation, and includes chain-terminating analogs.
- methylated nucleic acids e.g., 2′-O-methRNA
- peptide nucleic acids e.g., 2′-O-methRNA
- modified peptide nucleic acids e.g., locked nucleic acids
- any other structural moiety that can act substantially like a nucleotide or base, for example, by exhibiting base-complementarity with one or more bases that occur in DNA or RNA and/or being capable of base-complementary incorporation,
- Nucleotides for primer addition according to the invention preferably comprise a detectable label.
- Labeled nucleotides include any nucleotide that has been modified to include a label that is directly or indirectly detectable.
- Preferred labels include optically-detectable labels, including fluorescent labels or fluorophores, such as fluorescein, rhodamine, derivatized rhodamine dyes, such as TAMRA, phosphor, polymethadine dye, fluorescent phosphoramidite, Texas Red, green fluorescent protein, acridine, cyanine, cyanine 5 dye, cyanine 3 dye, 5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), BODIPY, 120 ALEXA or a derivative or modification of any of the foregoing, and also include such labeling systems as hapten labeling. Accordingly, methods of the invention further provide for exposing the primer/target nucleic acid duplex to a digoxi
- fluorescence resonance energy transfer is used to determine the base type incorporated into the primer. Fluorescence resonance energy transfer in the context of sequencing is described generally in Braslavasky et al., Sequence Information can be Obtained from Single DNA Molecules , Proc. Nat'l Acad. Sci., 100: 3960-3964 (2003), incorporated by reference herein.
- a donor fluorophore is attached to either the primer, polymerase, or template.
- Nucleotides added for incorporation into the primer comprise an acceptor fluorophore that is activated by the donor when the two are in proximity. Activation of the acceptor causes it to emit a characteristic wavelength of light and also quenches the donor.
- nucleotides labeled with a donor fluorophore also are useful in methods of the invention; FRET-based methods of the invention only require that a donor and acceptor fluorophore pair are used, a labeled nucleotide may comprise one fluorophore and either the template or the polymerase may comprise the other.
- labeling techniques result in a coincident fluorescent emission of the labels of the nucleotide and the labeled template or polymerase, or alternatively, the fluorescent emission of only one of the labels.
- the label is rendered undetectable by removing the label from the nucleotide or extended primer, neutralizing the label, or masking the label.
- methods according to the invention provide for neutralizing a label by photobleaching. This is accomplished by focusing a laser with a short laser pulse, for example, for a short duration of time with increasing laser intensity.
- a label is photocleaved.
- a light-sensitive label bound to a nucleotide is photocleaved by focusing a particular wavelength of light on the label.
- Labels also can be chemically cleaved. Labels may be removed from a substrate using reagents, such as NaOH or other appropriate buffer reagent.
- Preferred substrates include glass, silica, and others with the optical properties described herein.
- Surfaces for sequencing according to the invention may be coated with, for example, an epoxide, polytetrafluoroethylene or a derivative of polytetrafluoroethylene, such as silanized polytetrafluoroethylene, a polyelectrolyte multilayer (PEM), or the equivalent.
- PEM polyelectrolyte multilayer
- Primers useful in the invention hybridize to template in a manner that allows template-dependent sequencing-by-synthesis.
- the primer may comprise DNA, RNA or a mixture of both.
- the invention also teaches the use of stabilizing molecules used in connection with the primer or the primer/template duplex, such as locked nucleic acid or peptide nucleic acid analogs.
- the melting temperature of the primer/target nucleic acid duplex may be increased from about 3° to about 8° Celsius per PNA or LNA base included in the primer.
- the primer comprises a locked nucleic acid base on its 3′ terminus.
- the primer may comprise any portion of PNA or LNA bases, such as between about 10% and about 50%, more than about 50%, or less than about 10%, 20%, 30%, 40%, 50% or 60% of the total nucleic acid residues in the primer.
- the PNA or LNA bases may be consecutive in the primer or may be interspersed throughout the primer.
- PNA or LNA bases are spaced apart at a distance of at least one turn of the helix when the primer is hybridized to template.
- LNA or PNA analogs allows primers to be shorter than would be the case to achieve similar melting temperatures using conventional nucleic acids.
- the primer comprises fewer than 25 nucleic acids.
- Methods of the invention are suitable for de novo sequencing, re-sequencing, sequence analysis, DNA fingerprinting, polymorphism identification, for example single nucleotide polymorphisms (SNP) detection, as well as for research and clinical applications in genetics.
- methods according to the invention also identify alternate splice sites, enumerate copy number, measure gene expression, identify unknown RNA molecules present in cells at low copy number, annotate genomes by determining which sequences are actually transcribed, determine phylogenic relationships, and elucidate differentiation of cells.
- Methods and surfaces of the invention are useful in diagnostic, therapeutic, prognostic (including drug selection), and developmental applications.
- FIG. 1 depicts a dual biotinylation reaction securing a primer and target nucleic acid to a substrate.
- FIG. 2 depicts a primer and target nucleic acid biotinylated to align complementary nucleotides.
- FIG. 3 depicts three primers comprising locked nucleic acid bases.
- FIG. 4 depicts the structure of a locked nucleic acid base.
- FIG. 5 shows the relative stability of dual biotin duplex on a streptavidinated PEM.
- single molecule sequencing comprises repeated single base extension reactions followed by one or more wash steps. Sequencing occurs on single strands spaced apart such that each strand is individually optically resolvable. Spatial and temporal stability of the individual strands are important in order to preserve the integrity of the sequencing process.
- template/primer hybridization is a dynamic process. Primer melts off template at a low, but detectable rate. Once melting occurs, at least some portion of primer will be unavailable to re-anneal with template.
- Methods and surfaces of the invention address this problem by placing stabilizing binding partners on each of the template and primer and, optionally, utilizing stabilizing molecules that confer an increased melting temperature on the primer/template hybrid.
- a substrate may be made of any suitable material that allows single molecules to be individually optically resolvable.
- Substrates for use according to the invention can be two- or three-dimensional and can comprise a planar surface (e.g., a glass slide) or can be shaped.
- Appropriate substrates include glass (e.g., controlled pore glass (CPG)), quartz, plastic (such as polystyrene (low cross-linked and high cross-linked polystyrene), polycarbonate, polypropylene and poly(methymethacrylate)), acrylic copolymer, polyamide, silica, metal (e.g., alkanethiolate-derivatized gold), cellulose, nylon, latex, dextran, gel matrix (e.g., silica gel), polyacrolein, or composites.
- CPG controlled pore glass
- plastic such as polystyrene (low cross-linked and high cross-linked polystyrene), polycarbonate, polypropylene and poly(methymethacrylate)
- acrylic copolymer polyamide
- silica metal (e.g., alkanethiolate-derivatized gold)
- cellulose e.g., nylon, latex, dextran
- gel matrix e.
- a substrate used according to the invention includes a biocompatible or biologically inert material that is transparent to light and optically flat (i.e., with a minimal micro-roughness rating).
- Specially manufactured, or chemically derivatized, low background fluorescence substrates e.g., glass slides
- Substrates may be prepared and analyzed on either the top or bottom surface of the planar substrate (i.e., relative to the orientation of the substrate in the detection system).
- the invention also includes three-dimensional substrates such as spheres, tubes (e.g., capillary tubes), microwells, microfluidic devices, or any other structure suitable for anchoring a nucleic acid.
- a substrate can be a microparticle, a bead, a membrane, a slide, a plate, a micromachined chip, and the like.
- Substrates can include planar arrays or matrices capable of having regions that include populations of target nucleic acids or primers. Examples include nucleoside-derivatized CPG and polystyrene slides; derivatized magnetic slides; polystyrene grafted with polyethylene glycol; and the like.
- Factors for selecting substrates include, for example, the material, porosity, size, and shape. Other important factors to be considered in selecting appropriate substrates include size uniformity, efficiency as a synthesis support, and the substrate's optical properties, e.g., clear smooth substrates (free from defects) provide instrumentational advantages when detecting incorporation of nucleotides in single molecules (e.g., nucleic acids.).
- Substrates are coated with a surface that facilitates nucleic acid binding and that reduces background.
- Preferred coatings are epoxides, silanized epoxides, biotinylated epoxides, streptavidinated epoxides, polyelecrolyte multilayers, including those that are derivatized for nucleic acid attachment (e.g., biotinylated, streptavidinated, or coated with a binding partner on the template/primer.
- Surfaces used to attach duplexes according to the invention can be any surface to which a binding partner is capable of attaching.
- surfaces should be free of debris, especially debris capable of fluorescing.
- surfaces should be stable and transparent to light.
- Preferred surfaces are epoxy surfaces and polyelectrolyte multilayer surfaces. Either of those surfaces is easily derivatized as described in the art for attachment of binding pairs.
- epoxide surfaces are derivatized with silane or other species capable of receiving binding partners.
- binding pair members attached to template/primer hybrids attach directly to the surface via a molecule embedded in the surface that is not the normal binding partner for the binding pair member.
- Polyelectrolyte multilayer surfaces are formed from a variety of alternating layers of positive and negative charge. Preferred polyelectrolyte multilayer surfaces are described in detail below.
- a target nucleic acid for analysis may be obtained from a patient sample, e.g., from blood, urine, cerebrospinal fluid, seminal fluid, saliva, breast nipple aspirate, sputum, stool and biopsy tissue. Any tissue or body fluid specimen may be used according to methods of the invention.
- a target nucleic acid can come from a variety of sources.
- nucleic acids can be naturally occurring DNA or RNA isolated from any source, recombinant molecules, cDNA, or synthetic analogs, as known in the art.
- the target nucleic acid may be genomic DNA, genes, gene fragments, exons, introns, regulatory elements (such as promoters, enhancers, initiation and termination regions, expression regulatory factors, expression controls, and other control regions), DNA comprising one or more single-nucleotide polymorphisms (SNPs), allelic variants, and other mutations.
- SNPs single-nucleotide polymorphisms
- allelic variants allelic variants
- the full genome of one or more cells for example cells from different stages of diseases such as cancer.
- the target nucleic acid may also be mRNA, tRNA, rRNA, ribozymes, splice variants, antisense RNA, or siRNA.
- RNA with a recognition site for binding a polymerase, transcripts of a single cell, organelle or microorganism, and all or portions of RNA complements of one or more cells for example, cells from different stages of development or differentiation, and cells from different species.
- Nucleic acids can be obtained from any cell of a person, animal, plant, bacteria, or virus, including pathogenic microbes or other cellular organisms. Individual nucleic acids can be isolated for analysis.
- Methods of the invention also contemplate using a stabilizing molecule in a sequencing-by-synthesis reaction.
- the stabilizing molecule strengthens the primer/template bond and increases the specificity of incorporation of nucleotides into the primer, the melting temperature of the primer/target nucleic acid duplex, or both.
- a stabilizing molecule may comprise nucleotide or internucleotide analogs, or covalently bound minor groove binders or intercalators that enhance hybridization avidity or specificity of the primer to a target nucleic acid.
- the internucleotide analogs can comprise one or more of a phosphate ester analog such as alkyl phosphonates, phosphoroamidates, alkylphosphotriesters, phosphorothioates and phosphorodithioates.
- a phosphate ester analog such as alkyl phosphonates, phosphoroamidates, alkylphosphotriesters, phosphorothioates and phosphorodithioates.
- the stabilizing molecule comprises a minor groove binder.
- Minor groove binders are described in detail in U.S. Pat. No. 6,084,102 which is incorporated by reference in its entirety herein.
- a minor groove binder has a molecular weight of approximately 150 to approximately 2000 daltons, and typically covalently attaches to at least one of the nucleotides in a duplex. It incorporates into a duplex to strengthen the template/primer bond, thus increasing hybridization stability.
- a stabilizing molecule may comprise a conformationally restricted nucleotide analog such as a peptide nucleic acid base, a locked nucleic acid base or an oxetane modified base (for a discussion of base constraining oxetane modifications, see U.S. Published Patent Application No. 20040142946, the disclosure of which is incorporated by reference herein).
- a peptide nucleic acid is a nucleic acid analog in which the backbone comprises synthetic peptide like linkages (amide bonds) usually formed from N-(2-amino-ethyl)-glycine units, resulting in an achiral and uncharged molecule.
- PNA hybridizes with complementary nucleic acids with high affinity and specificity, and forms PNA/DNA and PNA/RNA duplexes having greater thermal and chemical stability than counterpart DNA/DNA duplexes.
- a locked nucleic acid is a bicyclic nucleic acid analog that contains one or more 2′-O, 4′-C methylene linkage(s), which effectively locks the furanose ring in a C3′-endo conformation.
- This methylene linkage “bridge” restricts the flexibility of the ribofuranose ring and locks the structure into a rigid bicyclic formation.
- locked nucleic acids demonstrate a much greater affinity and specificity to their complementary nucleic acids than do natural DNA counterparts and increases the thermal and chemical stability of a primer/target nucleic acid duplex. LNAs will hybridize to complementary nucleic acids even under adverse conditions, such as under low salt concentrations and in the presence of chaotropic agents.
- locked nucleic acids increase the melting point of the primer/target nucleic acid duplex by about 30 to about 8° Celsius per locked nucleic acid base incorporated in the primer.
- FIG. 4 shows an example of the structure of a locked nucleic acid base.
- the primer may comprise DNA, RNA or a mixture of both.
- Locked nucleic acid bases may be interspersed throughout a strand of a primer, as shown in FIG. 3 , or may be placed consecutively or singularly in predetermined locations. The amount and placement of the locked nucleic acid bases depends on the desired characteristics of the primer.
- the primer comprises a locked nucleic acid base at its 3′ terminus.
- the primer may comprise any portion of locked nucleic acid bases, such as between about 10% and about 50%, more than about 50%, or less than about 10%, 20%, 30%, 40%, 50% or 60% of the total bases in the primer.
- primer length is selected to facilitate hybridization to a sufficiently complementary region of the template nucleic acid downstream of the region to be analyzed.
- the exact lengths of the primers depend on many factors, including temperature and source of primer. Placement of locked nucleic acid bases throughout a primer allows for an increased melting temperature of the primer/target nucleic acid duplex during a sequencing reaction. This also allows the primer length to remain short compared to a primer that does not contain locked nucleic acid bases.
- Embodiments of this invention include primers with 20 bases or less, which incorporate from 1 to 12 or more locked nucleic acid bases. For example, a 20 base primer which includes 12 locked nucleic acid bases may yield a melting temperature of between about 80° to 90° C. According to one embodiment of the invention, the primer comprises less than about 30, 25, 20, 15, 10, or 5 bases.
- FIG. 3 shows three exemplary primers (DXS 17, 7G7A, and 377), each containing nine locked nucleic acids and comprising a sequence complementary to a known primer attachment site of a target nucleic acid. While FIG. 3 shows primers of known sequences complementary to a known region of a template, primers useful in the invention also include primers comprising a random sequences. Useful primers also include primers comprising a sequence that is complementary to a known priming region that has been ligated to a target nucleic acid.
- Primers can be synthetically made using conventional nucleic acid synthesis techniques. For example, primers are synthesized on an automated DNA synthesizer (e.g., Applied Biosystems, Inc., Foster City, Calif.) using standard chemistries, such as phosphoramidite chemistry, and the like. Alternative chemistries, e.g., resulting in non-natural backbone groups, such as phosphorothioate, phosphoramidate, and the like, may also be employed provided that, for example, the resulting oligonucleotides are compatible with the polymerizing agent.
- the primers can also be ordered commercially from a variety of companies which specialize in custom nucleic acids such as Operon, Inc. (Alameda, Calif.). Primers comprising locked nucleic acids are purchased commercially (Proligo® LLC, Boulder, Colo.) or prepared as needed by methods known in the art.
- the foregoing methods confer a significant advantage in single molecule reactions, in which one is tracking nucleotide incorporation into individual template/primer duplexes.
- Single molecule techniques provide the ability to observe discrete differences within and between individuals in terms of nucleotide sequence. Disruption of a hybrid impairs the ability to obtain full advantage from single molecule techniques because the loss of a hybrid represents the loss of significant information content relative to a bulk reaction in which there exist numerous copies of each hybrid pair. Methods of the invention maximize the ability to keep hybrid pairs intact and attached to substrate.
- annealing Conditions for hybridizing primers to target nucleic acids are known in the art.
- the annealing reaction is performed under conditions which are stringent enough to ensure sequence specificity, yet sufficiently permissive to allow formation of stable hybrids at an acceptable rate.
- the temperature and length of time required for primer annealing depend upon several factors including the base composition, length and concentration of the primer, and the nature of the solvent used, e.g., the concentration of cosolvents such as DMSO (dimethylsulfoxide), formamide, or glycerol, and counterions such as magnesium.
- hybridization annealing
- hybridization is carried out at a temperature that is approximately 5 to 10° Celsius below the melting temperature of the primer/target nucleic acid duplex in the annealing solvent. Annealing temperatures may be modified based on the amount of locked nucleic acid included in the primer, based on manufacturer's recommendation and methods known in the art.
- the primer/target nucleic acid duplex is exposed to a polymerase, and at least one nucleotide or nucleotide analog under conditions that allow for incorporation of the nucleotide into the primer.
- Polymerases useful in the invention include any polymerizing agent capable of catalyzing a template-dependant addition of a nucleotide to a primer, such as, Klenow, Vent ThermoSequenase®, 9° NTM, Therminator, Taq, Tfl, Tth, Tli, Pfu, and others.
- a thermophilic polymerase is used.
- the invention provides for the primer/target nucleic acid duplex to be exposed to the polymerase and nucleotide at a temperature between about 30° and about 80° Celsius, or at least about 50°, 60°, or 70° Celsius.
- Nucleotides useful in the invention include any nucleotide or nucleotide analog, whether naturally-occurring or synthetic.
- preferred nucleotides are adenine, cytosine, guanine, uracil, or thymine bases; xanthine or hypoxanthine, 5-bromouracil, 2-aminopurine, deoxyinosine, or methylated cytosine, such as 5-methylcytosine, and N4-methoxydeoxycytosine.
- bases of polynucleotide mimetics such as methylated nucleic acids, e.g., 2′-O-methRNA, peptide nucleic acids, modified peptide nucleic acids, locked nucleic acids, oxetane-modified bases and any other structural moiety that can act substantially like a nucleotide or base, for example, by exhibiting base-complementarity with one or more bases that occur in DNA or RNA and/or being capable of base-complementary incorporation, and includes chain-terminating analogs.
- Nucleotides particularly useful in the invention comprise detectable labels.
- Labeled nucleotides include any nucleotide that has been modified to include a label that is directly or indirectly detectable.
- Preferred labels include optically-detectable labels, including fluorescent labels or fluorophores, such as fluorescein, rhodamine, derivatized rhodamine dyes, such as TAMRA, phosphor, polymethadine dye, fluorescent phosphoramidite, Texas Red, green fluorescent protein, acridine, cyanine, cyanine 5 dye, cyanine 3 dye, 5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), BODIPY, 120 ALEXA or a derivative or modification of any of the foregoing, and also include such labeling systems as hapten labeling. Accordingly, methods of the invention further provide for exposing the primer/target nucleic acid duplex to a digoxigenin, a flu
- fluorescent labels include, but are not limited to, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; Brilliant Yellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5′′-dibromopyrogallol-sulfonaphthalein (Bromopyroga
- the label is rendered undetectable by removing the label from the nucleotide or extended primer, neutralizing the label, or masking the label.
- methods according to the invention provide for neutralizing a label by photobleaching. This is accomplished by focusing a laser with a short laser pulse, for example, for a short duration of time with increasing laser intensity.
- a label is photocleaved.
- a light-sensitive label bound to a nucleotide is photocleaved by focusing a particular wavelength of light on the label.
- Labels also can be chemically cleaved. Labels may be removed from a substrate using reagents, such as NaOH or other appropriate buffer reagent.
- the primer or the target nucleic acid can also include a detectable label.
- the label When the labeled primer and/or target nucleic acid are attached to the substrate, the label facilitates locating the bound molecule through imaging.
- the primer or target nucleic acid can be labeled with a fluorescent labeling moiety (e.g., Cy3 or Cy5), or any other means used to label nucleotides.
- the detectable label used to label the primer or target nucleic acid can be different from the label used on the nucleotides or nucleotide analogs in the subsequent extension reactions. Additionally, once the molecule has been localized, it may be desirable to render the label undetectable prior to the nucleotide incorporation detection steps by methods such as washing or photobleaching.
- a nucleotide analog according to the invention can be modified to remove, cap or modify the 3′ hydroxyl group.
- methods of the invention can include, for example, the step of removing the 3′ hydroxyl group from the incorporated nucleotide or nucleotide analog. By removing the 3′ hydroxyl group from the incorporated nucleotide in the primer, further extension is halted or impeded.
- the modified nucleotide can be engineered so that the 3′ hydroxyl group can be removed and/or added by chemical methods.
- a nucleotide analog can be modified to include a moiety that is sufficiently large to prevent or sterically hinder further chain elongation by interfering with the polymerase, thereby halting incorporation of additional nucleotides or nucleotide analogs. Subsequent removal of the moiety, or at least the steric-hindering portion of the moiety, can concomitantly reverse chain termination and allow chain elongation to proceed.
- the moiety also can be a label.
- chemically cleaving or photocleaving the blocking moiety may also chemically-bleach or photobleach the label, respectively.
- Incorporation of a nucleotide or a nucleotide analog and their locations on the surface of a substrate can be detected with single molecule sensitivity according to the invention.
- single molecule resolution is achieved by anchoring a target nucleic acid at a low concentration to a substrate, and then imaging nucleotide incorporation with for example, with total internal reflection fluorescence microscopy.
- a number of detection methods are available for use in single molecule analysis.
- Methods for visualizing single molecules within nucleic acids labeled with an intercalating dye include, for example, fluorescence microscopy. For example, the fluorescent spectrum and lifetime of a single molecule excited-state can be measured. Standard detectors such as a photomultiplier tube or avalanche photodiode can be used. Full field imaging with a two-stage image intensified COD camera also can be used. Additionally, low noise cooled CCD can also be used to detect single fluorescent molecules.
- the detection system for the signal may depend upon the labeling moiety used, which can be defined by the chemistry available.
- a combination of an optical fiber or CCD can be used in the detection step.
- the substrate is itself transparent to the radiation used, it is possible to have an incident light beam pass through the substrate with the detector located opposite the substrate from the primer.
- electromagnetic labels various forms of spectroscopy systems can be used.
- Various physical orientations for the detection system are available and known in the art.
- Optical systems include near-field scanning microscopy, far-field confocal microscopy, wide-field epi-illumination, light scattering, dark field microscopy, photoconversion, single and/or multiphoton excitation, spectral wavelength discrimination, fluorophore identification, evanescent wave illumination, and total internal reflection fluorescence (TIRF) microscopy.
- methods involve detection of laser-activated fluorescence using a microscope equipped with a camera, sometimes referred to as high-efficiency photon detection system.
- Suitable photon detection systems include, but are not limited to, photodiodes and intensified CCD cameras.
- an intensified charge couple device (ICCD) camera can be used.
- ICCD intensified charge couple device
- the use of an ICCD camera to image individual fluorescent dye molecules in a fluid near a surface provides numerous advantages. For example, with an ICCD optical setup, it is possible to acquire a sequence of images (movies) of fluorophores.
- biotin/avidin binding pairs When a biotin-streptavidin linkage is used to anchor a primer and a target nucleic acid to a substrate, the primer and target nucleic acid are biotinylated, while the surface of the substrate is coated with streptavidin. Because streptavidin is a tetramer, it is possible that both template and primer will bind to the same surface streptavidin. However, the dual biotin labels may bind to adjacent streptavidin molecules as well.
- the PEM surfaces were amine-derivatized by treatment with 10 mM NHS and 10 mM EDC in 0.1 M MES buffer, pH 6.0, 05M NaCl (coupling buffer) for 15 minutes. The surfaces were then rinsed twice in the coupling buffer and incubated for 1 hour in 0.1 mg/ml Streptavidin Plus (SA-20, Prozyme) in the coupling buffer.
- 10 mM NHS and 10 mM EDC in 0.1 M MES buffer, pH 6.0, 05M NaCl (coupling buffer) for 15 minutes.
- the surfaces were then rinsed twice in the coupling buffer and incubated for 1 hour in 0.1 mg/ml Streptavidin Plus (SA-20, Prozyme) in the coupling buffer.
- the resulting streptavidinated surfaces were rinsed in a 200 ⁇ l solution of dual-biotin duplex in which the primer had the sequence: 5′-Biotin-TEG-AA AAA CCC CTT ATG CAC TTA TCC TTT ACT (SEQ ID NO: 1) and the template had the sequence: 5′-TCA GCT GCG TCA GCT AGC GAC AGT AAA GGA TAA GTG CAT AAG GGG TTT TT -TEG-Biotin (SEQ ID NO: 2; primer binding region bolded and underlined).
- the terminal thymidine was labeled with cyanine-5 dye and the adenine two positions 5′ of the terminus was labeled with a cyanine-3 dye.
- the dual biotin duplexes referred to above were first bound to a streptavidinated PEM surface as described above. The surface was then exposed to 100 nM unlabeled biotin at 52° C. for 10 minutes. As a control, streptavidinated PEMs were incubated in parallel and then rinsed with dual-biotin duplex. All slides then were washed, imaged, and analyzed as described above. The data are shown in FIG. 5 . The results indicate that the dual-biotin duplexes on a streptavidinated PEM surface are surface-stable.
- Another experiment shows the stability of dual-biotin duplexes in single molecule sequencing.
- streptavidinated slides are prepared on a PEM as described above. The slides then are biotinylated. Fresh biotin-long chain polyethyloxide-amine (Biotin-LC-PEO, Pierce) is prepared in MES buffer (50 mg Biotin in 2.5 ml MES). A 5 ml aliquot of the EDC solution described above is combined with 5 ml of the Biotin-LC-PEO solution and diluted in MES buffer to a total volume of 96 ml by adding 86 ml of MES buffer to a 2.5 mM final EDC-biotin concentration.
- the PEM-coated slides are then immersed in that solution in a 100 ml beaker and incubated for 60 minutes at room temperature.
- the slides are then rinsed in MES buffer with gentle agitation for 10 seconds. Immersion and rinsing are repeated four times in clean 100 ml volumes. Slides are then incubated in the final bath for 10 minutes.
- the resulting biotin-coated slides are stored in Tris-NaCl buffer prior to streptavidination.
- Streptavidin-Plus (SA20, Prozyme) isdissolved in a solution of 10 mM NaCl buffer at 0.14 mg/ml and stirred 10 minutes at room temperature to thoroughly dissolve flakes. The resulting solution is filtered with a 0.22 ⁇ filter. Biotinylated slides described above are placed in this solution in a 100 ml beaker with a stir bar and stirred for 15 minutes at room temperature. The slides are then rinsed in 100 ml Tris-NaCl buffer with gentle agitation for 10 seconds. The rinse process is repeated 5 times in clean 100 ml volumes of 3 ⁇ SSC-0.1% Triton, incubating in the final bath for 10 minutes. Finally, the slides are transferred to a fresh bath of Tris-NaCl and agitated for 10 seconds. Slides arestored in Tris-NaCl buffer at 4° C. prior to use.
- 3′ bioinylated target nucleic acid templates 5′-TCA GCT GCG TCA GCT AGC GAC AGT AAA GGA TAA GTG CAT AAG GGG TTT TT-TEG-Biotin (SEQ ID NO: 2), are obtained from Integrated DNA Technologies (Coral, Iowa).
- 5′ biotinylated primers 5′-Biotin-TEG-AA AAA CCC CTT ATG CAC TTA TCC TTT ACT (SEQ ID NO: 1), comprising a cyanine-5 dye were hybridized to the templates and exposed to the streptavidinated surfaces described above at a concentration of 10 pM.
- the surfaces are washed with MES buffer and the surface was imaged using a Nikon TE-2000U upright microscope equipped with a total internal reflection objective (Nikon).
- the location of label represented the location of bound hybrid and the positions of label are noted. Positional detection can also be accomplished using unlabeled template/primer and adding labeled first base.
- nucleotide additions are accomplished using the Klenow fragment (exo-) polymerase (New England Biolabs) at 10 mM in Ecopol reaction buffer and a series of cyanine-labeled nucleotide triphosphates.
- an oxygen scavenging system is used (glucose (0.36%), glucose oxidase (8 U/ml), catalase (423 U/ml), Trolox (5 mM), Gallate (5 mM), DABCO (10 mM), and 2,4,6-octatrienoic acid).
- cyanine-5-labeled primer The positions of cyanine-5-labeled primer are recorded and bleached.
- dUTP-Cy3 in polymerase is added to the slides. If dUTP is incorporated into the primer, fluorescence resonance energy transfer (FRET) from the cyanine-5 on the primer will caus the cyanine-3 dye to emit and the location of the emission is detected.
- FRET fluorescence resonance energy transfer
- the cyanine-3 dye is kept unbleached and subsequent additions are with cyanine-5-labeled dNTPs, using FRET with cyanine-3 as the donor for detection of incorporation.
- FRET fluorescence resonance energy transfer
- a primer is designed to be complementary to a known primer attachment site of the target nucleic acid, and locked nucleic acid bases are substituted for certain nucleotides within the selected primer sequence. As many locked nucleic acid bases are selected as desired depending on the temperature and length of primer, up to a primer comprising 100% locked nucleic acids. The more locked nucleic acid substitutions into the primer, the greater the melting point of the primer/target nucleic acid duplex relative a primer of the same length lacking locked nucleic acid residues.
- FIG. 3 shows three primers synthesized with locked nucleic acids.
- DXS17, 7G7A, and 377 primers were synthesized as complementary strands to known regions of a template, each primer incorporating nine locked nucleic acids. When these primers are annealed to their respective templates, hybridization may be carried out at temperatures between about 80° C. to about 90° C.
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Abstract
Description
- The invention provides methods for sequencing a nucleic acid comprising stabilizing a primer/target nucleic acid duplex attached to a substrate. Generally, methods of the invention comprise the use of a dual-anchored primer/target nucleic acid duplex or stabilizing molecule.
- Completion of the human genome has paved the way for important insights into biologic structure and function. Knowledge of the human genome has given rise to inquiry into individual differences, as well as differences within an individual, as the basis for differences in biological function and dysfunction. For example, single nucleotide differences between individuals, called single nucleotide polymorphisms (SNPs), are responsible for dramatic phenotypic differences. Those differences can be outward expressions of phenotype or can involve the likelihood that an individual will get a specific disease or how that individual will respond to treatment. Moreover, subtle genomic changes have been shown to be responsible for the manifestation of genetic diseases, such as cancer. A true understanding of the duplexities in either normal or abnormal function will require large amounts of specific sequence information.
- An understanding of cancer also requires an understanding of genomic sequence duplexity. Cancer is a disease that is rooted in heterogeneous genomic instability. Most cancers develop from a series of genomic changes, some subtle and some significant, that occur in a small subpopulation of cells. Knowledge of the sequence variations that lead to cancer will lead to an understanding of the etiology of the disease, as well as ways to treat and prevent it. An essential first step in understanding genomic duplexity is the ability to perform high-resolution sequencing.
- Various approaches to nucleic acid sequencing exist. One conventional way to do bulk sequencing is by chain termination and gel separation, essentially as described by Sanger et al., Proc. Natl. Acad. Sci., 74(12): 5463-67 (1977). That method relies on the generation of a mixed population of nucleic acid fragments representing terminations at each base in a sequence. The fragments are then run on an electrophoretic gel and the sequence is revealed by the order of fragments in the gel. Another conventional bulk sequencing method relies on chemical degradation of nucleic acid fragments. See, Maxam et al., Proc. Natl. Acad. Sci., 74: 560-564 (1977). Finally, methods have been developed based upon sequencing by hybridization. See, e.g., Drrnanac, et al., Nature Biotech., 16: 54-58 (1998).
- Bulk sequencing techniques are not useful for the identification of subtle or rare nucleotide changes due to the many cloning, amplification and electrophoresis steps that complicate the process of gaining useful information regarding individual nucleotides. The ability to sequence and gain information from single molecules obtained from an individual patient is the next milestone for genomic sequencing. As such, research has evolved toward methods for rapid sequencing, such as single molecule sequencing technologies.
- There have been many proposals to develop new sequencing technologies based on single-molecule measurements, generally either by observing the interaction of particular proteins with DNA or by using ultra high resolution scanned probe microscopy. See, e.g., Rigler, et al., DNA-Sequencing at the Single Molecule Level, Journal of Biotechnology, 86(3): 161 (2001); Goodwin, P. M., et al., Application of Single Molecule Detection to DNA Sequencing. Nucleosides & Nucleotides, 16(5-6): 543-550 (1997); Howorka, S., et al., Sequence-Specific Detection of Individual DNA Strands using Engineered Nanopores, Nature Biotechnology, 19(7): 636-639 (2001); Meller, A., et al., Rapid Nanopore Discrimination Between Single Polynucleotide Molecules, Proceedings of the National Academy of Sciences of the United States of America, 97(3): 1079-1084 (2000); Driscoll, R. J., et al., Atomic-Scale Imaging of DNA Using Scanning Tunneling Microscopy. Nature, 346(6281): 294-296 (1990). Unlike conventional sequencing technologies, their speed and read-length would not be inherently limited by the resolving power of electrophoretic separation. Other methods proposed for single molecule sequencing include detecting individual nucleotides as they are incorporated into a primed template, i.e., sequencing by synthesis.
- While single molecule techniques have several advantages, implementation has been problematic. For example, the reproducibility and accuracy of many single molecule techniques rely upon the stability of a primer/target nucleic acid duplex attached to a solid substrate. However, incomplete binding of the primer to the template, disengagement of the primer from the template and disengagement of the duplex from the substrate are frequent occurrences in such single molecule techniques.
- Accordingly, there is a need in the art for methods and devices for sequencing generally, and single molecule sequencing in particular, including methods for stabilizing a target nucleic acid for sequence determination.
- The invention generally provides methods and surfaces for nucleic acid sequencing comprising stabilized primer/target nucleic acid duplexes on a surface. Methods of the invention generally contemplate the use of a primer/target nucleic acid duplex in which each of the primer and the template contain a molecule having a binding partner on the substrate. The primer/target is stabilized on the surface by binding of both the primer and the template to the surface. Binding pairs for use in the invention are any molecular pair that can be bound to a surface and attached to a nucleic acid. Some examples of preferred pairs include ligand/receptor, affinity pairs, antigen/antibody, and carbohydrate/lectin. For example, biotin/streptavidin, digoxigenin/anti-digoxigenin, and dinitrophenol/anti-dinitrophenol perform well in the invention. Other pairs are apparent to the skilled artisan based upon the description of the invention provided below.
- According to the invention, the primer contains a member of a binding pair at its 5′ terminus, and the template contains a member of a binding pair at its 3′ terminus or the primer contains a member of a binding pair at its 3′ terminus and the template contains a member of a binding pair at its 5′ terminus. Thus, the primer hybridizes to the template, and the two attached binding pair members are oriented to bind to their respective mates on the surface.
- The template and primer may contain the same type or species of binding pair or they may contain separate types or species. Binding may occur to a single species of binding partner or to separate members of the same species. For example, in one embodiment, both the template and the primer are bioinylated at opposite ends oriented to the surface (i.e., one at the 3′ end and one at the 5′ end) and the two biotin molecules adhere to the same streptavidin molecule (which has capacity to bind four biotins) on the surface. Alternatively, the two biotins adhere to separate streptavidin molecules spaced closely together on the surface. In another embodiment, the primer is attached to a member of a first binding pair and the template is attached to a member of a second binding pair. Upon hybridization, the first member attaches to its mate on the surface, and the second member attaches to its separate mate on the surface. In either embodiment, the combination of two separate mating pairs reduces loss of the hybrid due to either the template or the primer dissociating. It is apparent to the skilled artisan based upon this disclosure that any combination of binding pairs works to stabilize hybrid binding to a surface. For example, template and primer may have attached separate species of binder that, although distinct, bind to the same surface-bound mate.
- The invention comprises methods for sequencing nucleic acids using stabilized, support-bound primer/template hybrids as described above. In a preferred embodiment, methods of the invention comprise template-dependent sequencing by synthesis using a polymerase capable of adding nucleotides to the primer in a template-dependent fashion. The invention is particularly useful for single molecule nucleic acid sequencing in which primer/template duplex is attached to a substrate such that the duplex is individually optically resolvable. Individual strand sequence is determined by detecting ordered template-dependent nucleotide incorporation into the primer and compiling a sequence of the template based upon the order of incorporated nucleotides.
- The invention also provides for the use of a stabilizing molecule in template-dependent sequencing. Stabilizing molecules useful in the invention include, for example, locked nucleic acid (“LNA”) analogs and peptide nucleic acid (“PNA”) analogs. Generally, a stabilizing molecule increases the affinity and specificity of the primer/target nucleic acid bond, and increases the melting temperature of the primer/target nucleic acid duplex or the specificity of incorporation of a nucleotide into the primer in a sequencing by synthesis reaction. An example of a locked nucleic acid is shown in
FIG. 4 . Both locked nucleic acid and peptide nucleic acid analogs increase the melting temperature of the primer/template duplex and, therefore, confer stability on the hybrid, whether or not the anchoring strategies described above are used. - Polymerases useful in the invention include any polymerizing agent capable of catalyzing a template-dependent addition of a nucleotide or nucleotide analog to a primer. Depending on the characteristics of the target nucleic acid, a DNA polymerase, an RNA polymerase, or a reverse transcriptase can be used. According to one aspect of the invention, a thermophilic polymerase is used, such as ThermoSequenase®, 9° N™, Taq, Tfl, Tth, Tli, Therminator, or Pfu. In one embodiment, the invention provides for the primer/target nucleic acid duplex to be exposed to the polymerase and nucleotide at a temperature between about 30° and about 80° Celsius. A preferred polymerase is a Klenow fragment having reduced 3′-5′ exonuclease activity.
- Nucleotides useful in the invention include any nucleotide or nucleotide analog, whether naturally-occurring or synthetic. For example, preferred nucleotides are adenine, cytosine, guanine, uracil, or thymine bases; xanthine or hypoxanthine, 5-bromouracil, 2-aminopurine, deoxyinosine, or methylated cytosine, such as 5-methylcytosine, and N4-methoxydeoxycytosine. Also included are bases of polynucleotide mimetics, such as methylated nucleic acids, e.g., 2′-O-methRNA, peptide nucleic acids, modified peptide nucleic acids, locked nucleic acids and any other structural moiety that can act substantially like a nucleotide or base, for example, by exhibiting base-complementarity with one or more bases that occur in DNA or RNA and/or being capable of base-complementary incorporation, and includes chain-terminating analogs.
- Nucleotides for primer addition according to the invention preferably comprise a detectable label. Labeled nucleotides include any nucleotide that has been modified to include a label that is directly or indirectly detectable. Preferred labels include optically-detectable labels, including fluorescent labels or fluorophores, such as fluorescein, rhodamine, derivatized rhodamine dyes, such as TAMRA, phosphor, polymethadine dye, fluorescent phosphoramidite, Texas Red, green fluorescent protein, acridine, cyanine,
cyanine 5 dye,cyanine 3 dye, 5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), BODIPY, 120 ALEXA or a derivative or modification of any of the foregoing, and also include such labeling systems as hapten labeling. Accordingly, methods of the invention further provide for exposing the primer/target nucleic acid duplex to a digoxigenin, a fluorescein, an alkaline phosphatase or a peroxidase. - In one embodiment, fluorescence resonance energy transfer (FRET) is used to determine the base type incorporated into the primer. Fluorescence resonance energy transfer in the context of sequencing is described generally in Braslavasky et al., Sequence Information can be Obtained from Single DNA Molecules, Proc. Nat'l Acad. Sci., 100: 3960-3964 (2003), incorporated by reference herein. Essentially, in one embodiment, a donor fluorophore is attached to either the primer, polymerase, or template. Nucleotides added for incorporation into the primer comprise an acceptor fluorophore that is activated by the donor when the two are in proximity. Activation of the acceptor causes it to emit a characteristic wavelength of light and also quenches the donor. In this way, incorporation of a nucleotide in the primer sequence is detected by detection of acceptor emission. Of course, nucleotides labeled with a donor fluorophore also are useful in methods of the invention; FRET-based methods of the invention only require that a donor and acceptor fluorophore pair are used, a labeled nucleotide may comprise one fluorophore and either the template or the polymerase may comprise the other. Such labeling techniques result in a coincident fluorescent emission of the labels of the nucleotide and the labeled template or polymerase, or alternatively, the fluorescent emission of only one of the labels.
- In a preferred embodiment, after detection, the label is rendered undetectable by removing the label from the nucleotide or extended primer, neutralizing the label, or masking the label. In certain embodiments, methods according to the invention provide for neutralizing a label by photobleaching. This is accomplished by focusing a laser with a short laser pulse, for example, for a short duration of time with increasing laser intensity. In other embodiments, a label is photocleaved. For example, a light-sensitive label bound to a nucleotide is photocleaved by focusing a particular wavelength of light on the label. Generally, it may be preferable to use lasers having differing wavelengths for exciting and photocleaving. Labels also can be chemically cleaved. Labels may be removed from a substrate using reagents, such as NaOH or other appropriate buffer reagent.
- Preferred substrates include glass, silica, and others with the optical properties described herein. Surfaces for sequencing according to the invention may be coated with, for example, an epoxide, polytetrafluoroethylene or a derivative of polytetrafluoroethylene, such as silanized polytetrafluoroethylene, a polyelectrolyte multilayer (PEM), or the equivalent.
- Primers useful in the invention hybridize to template in a manner that allows template-dependent sequencing-by-synthesis. Depending on the target nucleic acid, the primer may comprise DNA, RNA or a mixture of both. The invention also teaches the use of stabilizing molecules used in connection with the primer or the primer/template duplex, such as locked nucleic acid or peptide nucleic acid analogs. According to the invention, the melting temperature of the primer/target nucleic acid duplex may be increased from about 3° to about 8° Celsius per PNA or LNA base included in the primer. In one embodiment, the primer comprises a locked nucleic acid base on its 3′ terminus. The primer may comprise any portion of PNA or LNA bases, such as between about 10% and about 50%, more than about 50%, or less than about 10%, 20%, 30%, 40%, 50% or 60% of the total nucleic acid residues in the primer. The PNA or LNA bases may be consecutive in the primer or may be interspersed throughout the primer. In a preferred embodiment, PNA or LNA bases are spaced apart at a distance of at least one turn of the helix when the primer is hybridized to template. The use of LNA or PNA analogs allows primers to be shorter than would be the case to achieve similar melting temperatures using conventional nucleic acids. According to one embodiment of the invention, the primer comprises fewer than 25 nucleic acids.
- Methods of the invention are suitable for de novo sequencing, re-sequencing, sequence analysis, DNA fingerprinting, polymorphism identification, for example single nucleotide polymorphisms (SNP) detection, as well as for research and clinical applications in genetics. Applied to RNA sequences, methods according to the invention also identify alternate splice sites, enumerate copy number, measure gene expression, identify unknown RNA molecules present in cells at low copy number, annotate genomes by determining which sequences are actually transcribed, determine phylogenic relationships, and elucidate differentiation of cells. Methods and surfaces of the invention are useful in diagnostic, therapeutic, prognostic (including drug selection), and developmental applications.
- As will be appreciated by one skilled in the art, individual features of the invention may be used separately or in any combination. A detailed description of embodiments of the invention is provided below. Other embodiments of the invention are apparent upon review of the detailed description that follows.
-
FIG. 1 depicts a dual biotinylation reaction securing a primer and target nucleic acid to a substrate. -
FIG. 2 depicts a primer and target nucleic acid biotinylated to align complementary nucleotides. -
FIG. 3 depicts three primers comprising locked nucleic acid bases. -
FIG. 4 depicts the structure of a locked nucleic acid base. -
FIG. 5 shows the relative stability of dual biotin duplex on a streptavidinated PEM. - The results of single molecule sequencing are influenced by the stability of substrate-bound primer/target nucleic acid duplexes. Typically, single molecule sequencing comprises repeated single base extension reactions followed by one or more wash steps. Sequencing occurs on single strands spaced apart such that each strand is individually optically resolvable. Spatial and temporal stability of the individual strands are important in order to preserve the integrity of the sequencing process. One way in which the spatial stability of a single molecule array can be disrupted is if the template and/or primer become disassociated with the surface. For example, template/primer hybridization is a dynamic process. Primer melts off template at a low, but detectable rate. Once melting occurs, at least some portion of primer will be unavailable to re-anneal with template. That is not necessarily a problem in a bulk sequencing reaction in which numerous copies of each template are available for sequencing. However, in single molecule sequencing, in which individual strands are sequenced, loss of any strand can have a significant effect on the result. Methods and surfaces of the invention address this problem by placing stabilizing binding partners on each of the template and primer and, optionally, utilizing stabilizing molecules that confer an increased melting temperature on the primer/template hybrid.
- I. General Considerations
- Substrates
- Generally, a substrate may be made of any suitable material that allows single molecules to be individually optically resolvable. Substrates for use according to the invention can be two- or three-dimensional and can comprise a planar surface (e.g., a glass slide) or can be shaped. Appropriate substrates include glass (e.g., controlled pore glass (CPG)), quartz, plastic (such as polystyrene (low cross-linked and high cross-linked polystyrene), polycarbonate, polypropylene and poly(methymethacrylate)), acrylic copolymer, polyamide, silica, metal (e.g., alkanethiolate-derivatized gold), cellulose, nylon, latex, dextran, gel matrix (e.g., silica gel), polyacrolein, or composites.
- Preferably, a substrate used according to the invention includes a biocompatible or biologically inert material that is transparent to light and optically flat (i.e., with a minimal micro-roughness rating). Specially manufactured, or chemically derivatized, low background fluorescence substrates (e.g., glass slides) are also contemplated according to the invention. Substrates may be prepared and analyzed on either the top or bottom surface of the planar substrate (i.e., relative to the orientation of the substrate in the detection system).
- The invention also includes three-dimensional substrates such as spheres, tubes (e.g., capillary tubes), microwells, microfluidic devices, or any other structure suitable for anchoring a nucleic acid. For example, a substrate can be a microparticle, a bead, a membrane, a slide, a plate, a micromachined chip, and the like. Substrates can include planar arrays or matrices capable of having regions that include populations of target nucleic acids or primers. Examples include nucleoside-derivatized CPG and polystyrene slides; derivatized magnetic slides; polystyrene grafted with polyethylene glycol; and the like.
- Factors for selecting substrates include, for example, the material, porosity, size, and shape. Other important factors to be considered in selecting appropriate substrates include size uniformity, efficiency as a synthesis support, and the substrate's optical properties, e.g., clear smooth substrates (free from defects) provide instrumentational advantages when detecting incorporation of nucleotides in single molecules (e.g., nucleic acids.).
- Substrates are coated with a surface that facilitates nucleic acid binding and that reduces background. Preferred coatings are epoxides, silanized epoxides, biotinylated epoxides, streptavidinated epoxides, polyelecrolyte multilayers, including those that are derivatized for nucleic acid attachment (e.g., biotinylated, streptavidinated, or coated with a binding partner on the template/primer.
- Surfaces
- Surfaces used to attach duplexes according to the invention can be any surface to which a binding partner is capable of attaching. For sequencing, surfaces should be free of debris, especially debris capable of fluorescing. Also, surfaces should be stable and transparent to light. Preferred surfaces are epoxy surfaces and polyelectrolyte multilayer surfaces. Either of those surfaces is easily derivatized as described in the art for attachment of binding pairs. For example, epoxide surfaces are derivatized with silane or other species capable of receiving binding partners. In certain embodiments, binding pair members attached to template/primer hybrids attach directly to the surface via a molecule embedded in the surface that is not the normal binding partner for the binding pair member. Polyelectrolyte multilayer surfaces are formed from a variety of alternating layers of positive and negative charge. Preferred polyelectrolyte multilayer surfaces are described in detail below.
- Target Nucleic Acids
- A target nucleic acid for analysis may be obtained from a patient sample, e.g., from blood, urine, cerebrospinal fluid, seminal fluid, saliva, breast nipple aspirate, sputum, stool and biopsy tissue. Any tissue or body fluid specimen may be used according to methods of the invention.
- A target nucleic acid can come from a variety of sources. For example, nucleic acids can be naturally occurring DNA or RNA isolated from any source, recombinant molecules, cDNA, or synthetic analogs, as known in the art. For example, the target nucleic acid may be genomic DNA, genes, gene fragments, exons, introns, regulatory elements (such as promoters, enhancers, initiation and termination regions, expression regulatory factors, expression controls, and other control regions), DNA comprising one or more single-nucleotide polymorphisms (SNPs), allelic variants, and other mutations. Also included is the full genome of one or more cells, for example cells from different stages of diseases such as cancer. The target nucleic acid may also be mRNA, tRNA, rRNA, ribozymes, splice variants, antisense RNA, or siRNA. Also contemplated according to the invention are RNA with a recognition site for binding a polymerase, transcripts of a single cell, organelle or microorganism, and all or portions of RNA complements of one or more cells, for example, cells from different stages of development or differentiation, and cells from different species. Nucleic acids can be obtained from any cell of a person, animal, plant, bacteria, or virus, including pathogenic microbes or other cellular organisms. Individual nucleic acids can be isolated for analysis.
- Stabilizing Molecules and Primers
- Methods of the invention also contemplate using a stabilizing molecule in a sequencing-by-synthesis reaction. The stabilizing molecule strengthens the primer/template bond and increases the specificity of incorporation of nucleotides into the primer, the melting temperature of the primer/target nucleic acid duplex, or both. A stabilizing molecule may comprise nucleotide or internucleotide analogs, or covalently bound minor groove binders or intercalators that enhance hybridization avidity or specificity of the primer to a target nucleic acid. The internucleotide analogs can comprise one or more of a phosphate ester analog such as alkyl phosphonates, phosphoroamidates, alkylphosphotriesters, phosphorothioates and phosphorodithioates.
- In one aspect, the stabilizing molecule comprises a minor groove binder. Minor groove binders are described in detail in U.S. Pat. No. 6,084,102 which is incorporated by reference in its entirety herein. Generally, a minor groove binder has a molecular weight of approximately 150 to approximately 2000 daltons, and typically covalently attaches to at least one of the nucleotides in a duplex. It incorporates into a duplex to strengthen the template/primer bond, thus increasing hybridization stability.
- In one embodiment, a stabilizing molecule may comprise a conformationally restricted nucleotide analog such as a peptide nucleic acid base, a locked nucleic acid base or an oxetane modified base (for a discussion of base constraining oxetane modifications, see U.S. Published Patent Application No. 20040142946, the disclosure of which is incorporated by reference herein). A peptide nucleic acid is a nucleic acid analog in which the backbone comprises synthetic peptide like linkages (amide bonds) usually formed from N-(2-amino-ethyl)-glycine units, resulting in an achiral and uncharged molecule. PNA hybridizes with complementary nucleic acids with high affinity and specificity, and forms PNA/DNA and PNA/RNA duplexes having greater thermal and chemical stability than counterpart DNA/DNA duplexes.
- A locked nucleic acid is a bicyclic nucleic acid analog that contains one or more 2′-O, 4′-C methylene linkage(s), which effectively locks the furanose ring in a C3′-endo conformation. This methylene linkage “bridge” restricts the flexibility of the ribofuranose ring and locks the structure into a rigid bicyclic formation. Because of its unique structural conformation, locked nucleic acids demonstrate a much greater affinity and specificity to their complementary nucleic acids than do natural DNA counterparts and increases the thermal and chemical stability of a primer/target nucleic acid duplex. LNAs will hybridize to complementary nucleic acids even under adverse conditions, such as under low salt concentrations and in the presence of chaotropic agents. According to one aspect of the invention, locked nucleic acids increase the melting point of the primer/target nucleic acid duplex by about 30 to about 8° Celsius per locked nucleic acid base incorporated in the primer.
FIG. 4 shows an example of the structure of a locked nucleic acid base. - Depending on the target nucleic acid, the primer may comprise DNA, RNA or a mixture of both. Locked nucleic acid bases may be interspersed throughout a strand of a primer, as shown in
FIG. 3 , or may be placed consecutively or singularly in predetermined locations. The amount and placement of the locked nucleic acid bases depends on the desired characteristics of the primer. In one embodiment, the primer comprises a locked nucleic acid base at its 3′ terminus. The primer may comprise any portion of locked nucleic acid bases, such as between about 10% and about 50%, more than about 50%, or less than about 10%, 20%, 30%, 40%, 50% or 60% of the total bases in the primer. - In general, primer length is selected to facilitate hybridization to a sufficiently complementary region of the template nucleic acid downstream of the region to be analyzed. The exact lengths of the primers depend on many factors, including temperature and source of primer. Placement of locked nucleic acid bases throughout a primer allows for an increased melting temperature of the primer/target nucleic acid duplex during a sequencing reaction. This also allows the primer length to remain short compared to a primer that does not contain locked nucleic acid bases. Embodiments of this invention include primers with 20 bases or less, which incorporate from 1 to 12 or more locked nucleic acid bases. For example, a 20 base primer which includes 12 locked nucleic acid bases may yield a melting temperature of between about 80° to 90° C. According to one embodiment of the invention, the primer comprises less than about 30, 25, 20, 15, 10, or 5 bases.
-
FIG. 3 shows three exemplary primers (DXS 17, 7G7A, and 377), each containing nine locked nucleic acids and comprising a sequence complementary to a known primer attachment site of a target nucleic acid. WhileFIG. 3 shows primers of known sequences complementary to a known region of a template, primers useful in the invention also include primers comprising a random sequences. Useful primers also include primers comprising a sequence that is complementary to a known priming region that has been ligated to a target nucleic acid. - Primers can be synthetically made using conventional nucleic acid synthesis techniques. For example, primers are synthesized on an automated DNA synthesizer (e.g., Applied Biosystems, Inc., Foster City, Calif.) using standard chemistries, such as phosphoramidite chemistry, and the like. Alternative chemistries, e.g., resulting in non-natural backbone groups, such as phosphorothioate, phosphoramidate, and the like, may also be employed provided that, for example, the resulting oligonucleotides are compatible with the polymerizing agent. The primers can also be ordered commercially from a variety of companies which specialize in custom nucleic acids such as Operon, Inc. (Alameda, Calif.). Primers comprising locked nucleic acids are purchased commercially (Proligo® LLC, Boulder, Colo.) or prepared as needed by methods known in the art.
- The foregoing methods confer a significant advantage in single molecule reactions, in which one is tracking nucleotide incorporation into individual template/primer duplexes. Single molecule techniques provide the ability to observe discrete differences within and between individuals in terms of nucleotide sequence. Disruption of a hybrid impairs the ability to obtain full advantage from single molecule techniques because the loss of a hybrid represents the loss of significant information content relative to a bulk reaction in which there exist numerous copies of each hybrid pair. Methods of the invention maximize the ability to keep hybrid pairs intact and attached to substrate.
- Primer Hybridization
- Conditions for hybridizing primers to target nucleic acids are known in the art. The annealing reaction is performed under conditions which are stringent enough to ensure sequence specificity, yet sufficiently permissive to allow formation of stable hybrids at an acceptable rate. The temperature and length of time required for primer annealing depend upon several factors including the base composition, length and concentration of the primer, and the nature of the solvent used, e.g., the concentration of cosolvents such as DMSO (dimethylsulfoxide), formamide, or glycerol, and counterions such as magnesium. Typically, hybridization (annealing) is carried out at a temperature that is approximately 5 to 10° Celsius below the melting temperature of the primer/target nucleic acid duplex in the annealing solvent. Annealing temperatures may be modified based on the amount of locked nucleic acid included in the primer, based on manufacturer's recommendation and methods known in the art.
- Primer Extension and Labeling
- During primer extension, the primer/target nucleic acid duplex is exposed to a polymerase, and at least one nucleotide or nucleotide analog under conditions that allow for incorporation of the nucleotide into the primer. Polymerases useful in the invention include any polymerizing agent capable of catalyzing a template-dependant addition of a nucleotide to a primer, such as, Klenow, Vent ThermoSequenase®, 9° N™, Therminator, Taq, Tfl, Tth, Tli, Pfu, and others. According to one aspect of the invention, a thermophilic polymerase is used. In one embodiment, the invention provides for the primer/target nucleic acid duplex to be exposed to the polymerase and nucleotide at a temperature between about 30° and about 80° Celsius, or at least about 50°, 60°, or 70° Celsius.
- Nucleotides useful in the invention include any nucleotide or nucleotide analog, whether naturally-occurring or synthetic. For example, preferred nucleotides are adenine, cytosine, guanine, uracil, or thymine bases; xanthine or hypoxanthine, 5-bromouracil, 2-aminopurine, deoxyinosine, or methylated cytosine, such as 5-methylcytosine, and N4-methoxydeoxycytosine. Also included are bases of polynucleotide mimetics, such as methylated nucleic acids, e.g., 2′-O-methRNA, peptide nucleic acids, modified peptide nucleic acids, locked nucleic acids, oxetane-modified bases and any other structural moiety that can act substantially like a nucleotide or base, for example, by exhibiting base-complementarity with one or more bases that occur in DNA or RNA and/or being capable of base-complementary incorporation, and includes chain-terminating analogs.
- Nucleotides particularly useful in the invention comprise detectable labels. Labeled nucleotides include any nucleotide that has been modified to include a label that is directly or indirectly detectable. Preferred labels include optically-detectable labels, including fluorescent labels or fluorophores, such as fluorescein, rhodamine, derivatized rhodamine dyes, such as TAMRA, phosphor, polymethadine dye, fluorescent phosphoramidite, Texas Red, green fluorescent protein, acridine, cyanine,
cyanine 5 dye,cyanine 3 dye, 5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), BODIPY, 120 ALEXA or a derivative or modification of any of the foregoing, and also include such labeling systems as hapten labeling. Accordingly, methods of the invention further provide for exposing the primer/target nucleic acid duplex to a digoxigenin, a fluorescein, an alkaline phosphatase or a peroxidase. - Other suitable fluorescent labels include, but are not limited to, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; Brilliant Yellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride); 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin and derivatives; eosin, eosin isothiocyanate, erythrosin and derivatives; erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein derivatives; 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine; tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid; terbium chelate derivatives; Cy5.5; Cy7; IRD 700; IRD 800; La Jolta Blue; phthalo cyanine; and naphthalo cyanine.
- In a preferred embodiment, after detection, the label is rendered undetectable by removing the label from the nucleotide or extended primer, neutralizing the label, or masking the label. In certain embodiments, methods according to the invention provide for neutralizing a label by photobleaching. This is accomplished by focusing a laser with a short laser pulse, for example, for a short duration of time with increasing laser intensity. In other embodiments, a label is photocleaved. For example, a light-sensitive label bound to a nucleotide is photocleaved by focusing a particular wavelength of light on the label. Generally, it may be preferable to use lasers having differing wavelengths for exciting and photocleaving. Labels also can be chemically cleaved. Labels may be removed from a substrate using reagents, such as NaOH or other appropriate buffer reagent.
- Further, the primer or the target nucleic acid can also include a detectable label. When the labeled primer and/or target nucleic acid are attached to the substrate, the label facilitates locating the bound molecule through imaging. The primer or target nucleic acid can be labeled with a fluorescent labeling moiety (e.g., Cy3 or Cy5), or any other means used to label nucleotides. The detectable label used to label the primer or target nucleic acid can be different from the label used on the nucleotides or nucleotide analogs in the subsequent extension reactions. Additionally, once the molecule has been localized, it may be desirable to render the label undetectable prior to the nucleotide incorporation detection steps by methods such as washing or photobleaching.
- A nucleotide analog according to the invention can be modified to remove, cap or modify the 3′ hydroxyl group. As such, in certain embodiments, methods of the invention can include, for example, the step of removing the 3′ hydroxyl group from the incorporated nucleotide or nucleotide analog. By removing the 3′ hydroxyl group from the incorporated nucleotide in the primer, further extension is halted or impeded. In certain embodiments, the modified nucleotide can be engineered so that the 3′ hydroxyl group can be removed and/or added by chemical methods. Alternatively, a nucleotide analog can be modified to include a moiety that is sufficiently large to prevent or sterically hinder further chain elongation by interfering with the polymerase, thereby halting incorporation of additional nucleotides or nucleotide analogs. Subsequent removal of the moiety, or at least the steric-hindering portion of the moiety, can concomitantly reverse chain termination and allow chain elongation to proceed. In some embodiments, the moiety also can be a label. As such, in those embodiments, chemically cleaving or photocleaving the blocking moiety may also chemically-bleach or photobleach the label, respectively.
- Detection of Incorporated Nucleotides
- Incorporation of a nucleotide or a nucleotide analog and their locations on the surface of a substrate can be detected with single molecule sensitivity according to the invention. In some aspects of the invention, single molecule resolution is achieved by anchoring a target nucleic acid at a low concentration to a substrate, and then imaging nucleotide incorporation with for example, with total internal reflection fluorescence microscopy.
- A number of detection methods are available for use in single molecule analysis. Methods for visualizing single molecules within nucleic acids labeled with an intercalating dye include, for example, fluorescence microscopy. For example, the fluorescent spectrum and lifetime of a single molecule excited-state can be measured. Standard detectors such as a photomultiplier tube or avalanche photodiode can be used. Full field imaging with a two-stage image intensified COD camera also can be used. Additionally, low noise cooled CCD can also be used to detect single fluorescent molecules.
- The detection system for the signal may depend upon the labeling moiety used, which can be defined by the chemistry available. For optical signals, a combination of an optical fiber or CCD can be used in the detection step. In the embodiments where the substrate is itself transparent to the radiation used, it is possible to have an incident light beam pass through the substrate with the detector located opposite the substrate from the primer. For electromagnetic labels, various forms of spectroscopy systems can be used. Various physical orientations for the detection system are available and known in the art.
- A number of approaches can be used to detect incorporation of fluorescently-labeled nucleotides into a single molecule. Optical systems include near-field scanning microscopy, far-field confocal microscopy, wide-field epi-illumination, light scattering, dark field microscopy, photoconversion, single and/or multiphoton excitation, spectral wavelength discrimination, fluorophore identification, evanescent wave illumination, and total internal reflection fluorescence (TIRF) microscopy. In general, methods involve detection of laser-activated fluorescence using a microscope equipped with a camera, sometimes referred to as high-efficiency photon detection system. Suitable photon detection systems include, but are not limited to, photodiodes and intensified CCD cameras. For example, as illustrated in
FIG. 2 , an intensified charge couple device (ICCD) camera can be used. The use of an ICCD camera to image individual fluorescent dye molecules in a fluid near a surface provides numerous advantages. For example, with an ICCD optical setup, it is possible to acquire a sequence of images (movies) of fluorophores. - Certain embodiments of the invention are described in the following examples, which are not meant to be limiting.
- General methods of the invention were demonstrated using biotin/avidin binding pairs. When a biotin-streptavidin linkage is used to anchor a primer and a target nucleic acid to a substrate, the primer and target nucleic acid are biotinylated, while the surface of the substrate is coated with streptavidin. Because streptavidin is a tetramer, it is possible that both template and primer will bind to the same surface streptavidin. However, the dual biotin labels may bind to adjacent streptavidin molecules as well.
- Two experiments were done to determine the binding stability of the dual biotin constructs. A first experiment was conducted in order to determine the stability of dual biotin duplex on a polyelectrolyte multilayer (PEM) surface. This experiment was done using covalent streptavidin attachment to a PEM surface. The PEM surfaces were prepared as follows. Polyethyleneimine (PEI) and pollyallylamine (PAA, Sigma, St. Louis, Mo.) were dissolved separately by stirring in MilliQ water and the pH was adjusted to 8.0 with dilute HCl. The solutions were filtered using a 0.22μ filter flask and stored at 4° C. Clean glass slides were then alternatively immersed for 10 minutes in the PEI and PAA solutions four times each with an 8 minute rinse using MilliQ water between each immersion. After the last rinse, the slides were kept immersed in water. The slides were then transferred to MES buffer (2-[N-morpholino] ethanesulfonic acid), pH 5.5 for EDC-induced crosslinking of the PEM. A 10 mM solution of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) was prepared in MES buffer, filtered and added to the solution containing the PEM-coated slides for 1 hour at room temperature. Slides were then rinsed in MES buffer and stored.
- Next, the PEM surfaces were amine-derivatized by treatment with 10 mM NHS and 10 mM EDC in 0.1 M MES buffer, pH 6.0, 05M NaCl (coupling buffer) for 15 minutes. The surfaces were then rinsed twice in the coupling buffer and incubated for 1 hour in 0.1 mg/ml Streptavidin Plus (SA-20, Prozyme) in the coupling buffer. The resulting streptavidinated surfaces were rinsed in a 200 μl solution of dual-biotin duplex in which the primer had the sequence: 5′-Biotin-TEG-AA AAA CCC CTT ATG CAC TTA TCC TTT ACT (SEQ ID NO: 1) and the template had the sequence: 5′-TCA GCT GCG TCA GCT AGC GAC AGT AAA GGA TAA GTG CAT AAG GGG TTT TT-TEG-Biotin (SEQ ID NO: 2; primer binding region bolded and underlined). At its 3′ end, the terminal thymidine was labeled with cyanine-5 dye and the adenine two
positions 5′ of the terminus was labeled with a cyanine-3 dye. Surfaces were soaked for 5 minutes and then rinsed once in 20 mM Tris, pH 8.0, 50 mM NaCl, 0.01% Triton (Rinse Buffer). The surfaces were then rinsed 5 times in 3×SSC-0.1% Triton, with a 10 minute soak in the last rinse. Finally, the surfaces were rinsed in two changes of the Rinse Buffer. The resulting surface had dual-biotin primer/target nucleic acid duplex bound to streptavidin. A second set of slides was rinsed with the dual biotin duplexes as described above, but was also challenged with 100 nM unlabeled biotin. All slides were imaged over a 1000μ2 area and analyzed in ImagePro (Media Cybernetics, San Diego) using a dark image background subtraction algorithm. - Visualization of surface-bound duplexes was accomplished using fluorescence resonance energy transfer (FRET), with the cyanine-5 labeled thymidine as the donor and the cyanine-3 labeled adenine as the acceptor. Slides were placed on a Nikon Eclipse TE-2000 inverted microscope with a total internal reflection objective. The dual-biotin duplex slides showed 309.7 counts/pixel and the biotin-challenged slides showed 10.1 counts/pixel. These results indicate that the dual-biotin duplexes were binding to streptavidin on the PEM in a specific manner, as the cold biotin was able to compete away duplex binding.
- In a separate experiment, the dual biotin duplexes referred to above were first bound to a streptavidinated PEM surface as described above. The surface was then exposed to 100 nM unlabeled biotin at 52° C. for 10 minutes. As a control, streptavidinated PEMs were incubated in parallel and then rinsed with dual-biotin duplex. All slides then were washed, imaged, and analyzed as described above. The data are shown in
FIG. 5 . The results indicate that the dual-biotin duplexes on a streptavidinated PEM surface are surface-stable. - Another experiment shows the stability of dual-biotin duplexes in single molecule sequencing. For this experiment, streptavidinated slides are prepared on a PEM as described above. The slides then are biotinylated. Fresh biotin-long chain polyethyloxide-amine (Biotin-LC-PEO, Pierce) is prepared in MES buffer (50 mg Biotin in 2.5 ml MES). A 5 ml aliquot of the EDC solution described above is combined with 5 ml of the Biotin-LC-PEO solution and diluted in MES buffer to a total volume of 96 ml by adding 86 ml of MES buffer to a 2.5 mM final EDC-biotin concentration. The PEM-coated slides are then immersed in that solution in a 100 ml beaker and incubated for 60 minutes at room temperature. The slides are then rinsed in MES buffer with gentle agitation for 10 seconds. Immersion and rinsing are repeated four times in clean 100 ml volumes. Slides are then incubated in the final bath for 10 minutes. The resulting biotin-coated slides are stored in Tris-NaCl buffer prior to streptavidination.
- Streptavidin-Plus (SA20, Prozyme) isdissolved in a solution of 10 mM NaCl buffer at 0.14 mg/ml and stirred 10 minutes at room temperature to thoroughly dissolve flakes. The resulting solution is filtered with a 0.22μ filter. Biotinylated slides described above are placed in this solution in a 100 ml beaker with a stir bar and stirred for 15 minutes at room temperature. The slides are then rinsed in 100 ml Tris-NaCl buffer with gentle agitation for 10 seconds. The rinse process is repeated 5 times in clean 100 ml volumes of 3×SSC-0.1% Triton, incubating in the final bath for 10 minutes. Finally, the slides are transferred to a fresh bath of Tris-NaCl and agitated for 10 seconds. Slides arestored in Tris-NaCl buffer at 4° C. prior to use.
- 3′ bioinylated target nucleic acid templates: 5′-TCA GCT GCG TCA GCT AGC GAC AGT AAA GGA TAA GTG CAT AAG GGG TTT TT-TEG-Biotin (SEQ ID NO: 2), are obtained from Integrated DNA Technologies (Coral, Iowa). 5′ biotinylated primers: 5′-Biotin-TEG-AA AAA CCC CTT ATG CAC TTA TCC TTT ACT (SEQ ID NO: 1), comprising a cyanine-5 dye were hybridized to the templates and exposed to the streptavidinated surfaces described above at a concentration of 10 pM. After incubation for 10 minutes, the surfaces are washed with MES buffer and the surface was imaged using a Nikon TE-2000U upright microscope equipped with a total internal reflection objective (Nikon). The location of label represented the location of bound hybrid and the positions of label are noted. Positional detection can also be accomplished using unlabeled template/primer and adding labeled first base. To determine the stability of bound duplexes, nucleotide additions are accomplished using the Klenow fragment (exo-) polymerase (New England Biolabs) at 10 mM in Ecopol reaction buffer and a series of cyanine-labeled nucleotide triphosphates. To reduce bleaching of the fluorescent dyes, an oxygen scavenging system is used (glucose (0.36%), glucose oxidase (8 U/ml), catalase (423 U/ml), Trolox (5 mM), Gallate (5 mM), DABCO (10 mM), and 2,4,6-octatrienoic acid).
- The positions of cyanine-5-labeled primer are recorded and bleached. dUTP-Cy3 in polymerase is added to the slides. If dUTP is incorporated into the primer, fluorescence resonance energy transfer (FRET) from the cyanine-5 on the primer will caus the cyanine-3 dye to emit and the location of the emission is detected. The cyanine-3 dye is kept unbleached and subsequent additions are with cyanine-5-labeled dNTPs, using FRET with cyanine-3 as the donor for detection of incorporation. The results show that dual-biotin duplex is a stable template for template-dependent sequencing.
- The skilled artisan understands that there are numerous other embodiments of the invention in terms of surfaces, binding partners and the like that can be manipulated in order to achieve the stability results shown above.
- A primer is designed to be complementary to a known primer attachment site of the target nucleic acid, and locked nucleic acid bases are substituted for certain nucleotides within the selected primer sequence. As many locked nucleic acid bases are selected as desired depending on the temperature and length of primer, up to a primer comprising 100% locked nucleic acids. The more locked nucleic acid substitutions into the primer, the greater the melting point of the primer/target nucleic acid duplex relative a primer of the same length lacking locked nucleic acid residues.
-
FIG. 3 shows three primers synthesized with locked nucleic acids. DXS17, 7G7A, and 377 primers were synthesized as complementary strands to known regions of a template, each primer incorporating nine locked nucleic acids. When these primers are annealed to their respective templates, hybridization may be carried out at temperatures between about 80° C. to about 90° C. - The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
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