Patent Application
20090099027
This application relates generally to methods of modifying surfaces for the immobilization of particles.
Nucleic acid sequencing techniques are widely employed in basic research. In addition, sequencing techniques are becoming increasingly important in clinical diagnosis. For example, diagnostic tests based upon particular sequence variations are already in use for a variety of different diseases. Data obtained from nucleic acid sequencing can be used to determine if a particular polynucleotide differs in sequence from a reference polynucleotide. Sequencing data can also be used to confirm the presence of a particular polynucleotide sequence in a sample, determine partial sequence information and determine the identity and order of nucleotides within a polynucleotide.
There still exists a need for more rapid and accurate methods of sequence determination.
A method is provided that comprises:
reacting a nucleophilic group on the surface of a substrate with a molecule comprising a plurality of electrophilic groups thereby providing one or more free electrophilic groups on the surface of the substrate; and
reacting nucleophilic groups on a surface of a particulate material with the one or more free electrophilic groups on the surface of the substrate to covalently attach the particulate material to the substrate.
An article of manufacture is also provided that comprises:
a particulate material comprising surface functional groups;
a support comprising surface functional groups;
wherein surface functional groups of the particulate material are covalently attached to surface functional groups on the support via a linker group comprising the moiety:
A method is also provided that comprises:
reacting a nucleophilic group on the surface of a substrate with the compound represented by the formula:
or a polymer having a moiety represented by the formula:
An article of manufacture is also provided that comprises a moiety covalently attached to a support surface, wherein the moiety is represented by the formula:
wherein R1 represents a linking group and “SUPPORT” represents the support surface; or
wherein n is a positive integer, “SUPPORT” represent the support surface, R2 is a first chemical group, R3 is a second chemical group and R4 is a linker group.
A method is also provided that comprises:
(a) hybridizing an initializing oligonucleotide probe to a target polynucleotide to form a probe-target duplex, wherein the oligonucleotide probe has an extendable probe terminus, wherein the target polynucleotide is attached to a particulate material and wherein the particulate material is covalently attached to the surface of a support;
(b) ligating a first end of an extension oligonucleotide probe to the extendable probe terminus thereby forming an extended duplex containing an extended oligonucleotide probe, wherein the extension oligonucleotide probe comprises a cleavage site and a detectable label;
(c) identifying one or more nucleotides in the target polynucleotide by detecting the label attached to the just-ligated extension oligonucleotide probe;
(d) cleaving the just-ligated extension oligonucleotide probe at the cleavage site to generate the extendable probe terminus, wherein cleavage removes a portion of the just-ligated extension oligonucleotide probe that comprises the label from the probe-target duplex; and
(e) repeating steps (b), (c) and (d).
A method of sequencing a nucleic acid is also provided which comprises:
(a) hybridizing a primer to a target polynucleotide to form a primer-target duplex, wherein the target polynucleotide is attached to a particulate material at a 5′ end and wherein the particulate material is covalently attached to the surface of a support;
(b) contacting the primer-target duplex with a polymerase and one or more different nucleotide analogs to incorporate a nucleotide analog onto the 3′ end of the primer thereby forming an extended primer strand, wherein the incorporated nucleotide analog terminates the polymerase reaction and wherein each of the one or more nucleotide analogs comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine and uracil and their analogs (ii) a unique label attached to the base or analog thereof via a cleavable linker; (iii) a deoxyribose; and (iv) a cleavable chemical group which caps an —OH group at a 3′-position of the deoxyribose;
(c) washing the surface of the support to remove any unincorporated nucleotide analogs;
(d) detecting the unique label attached to the just-incorporated nucleotide analog to thereby identify the just-incorporated nucleotide analog;
(e) optionally, permanently capping any unreacted —OH group on the extended primer strand;
(f) cleaving the cleavable linker between the just incorporated nucleotide analog and the unique label;
(g) cleaving the chemical group capping the —OH group at the 3′-position of the deoxyribose of the just incorporated nucleotide analog to uncap the —OH group;
(h) washing the surface of the support to remove cleaved compounds;
(i) repeating steps (b)-(h).
These and other features of the present teachings are set forth herein.
The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in interpreting the document where the term is originally used). The use of “or” herein means “and/or” unless stated otherwise or where the use of “and/or” is clearly inappropriate. The use of “a” herein means “one or more” unless stated otherwise or where the use of “one or more” is clearly inappropriate. The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of” and/or “consisting of.”
As used herein, the term “nucleoside” includes 2′ deoxy nucleosides and 2′-hydroxyl nucleosides. The term “analogs” in reference to nucleosides includes synthetic nuceosides having modified base moieties and/or modified sugar moieties. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce degeneracy, increase specificity, and the like.
As used herein, the phrase “nucleotide analog” refers to a chemical compound that is structurally and functionally similar to a nucleotide and which can be recognized by a polymerase as a substrate. Nucleotide analogs include nucleotides comprising labels attached to the nucleotide via a cleavable linker and nucleotides in which the —OH group at the 3′ position of the deoxyribose is capped (e.g., with a chemical moiety such as —CH2OCH3 or —CH2CH═CH2). Nucleotide analogs of this type are disclosed in U.S. Pat. No. 6,664,079 B2.
As used herein, to cap (or capping) an —OH group means to replace the hydrogen of the —OH group with a different chemical group. The —OH group can be capped with a cleavable chemical group. To uncap (or uncapping) means to cleave the chemical group from a capped —OH group and to replace the chemical group with “H”. Suitable means of capping and uncapping —OH groups are disclosed in U.S. Pat. No. 6,664,079 B2.
As used herein, the phrase “oligonucleotide” refers to a linear oligomer of nucleosides or analogs thereof, including deoxyribonucleosides, ribonucleosides and the like. Oligonucleotides can range in size from a few monomeric units (e.g., 3 to 4 units) to several hundred monomeric units.
As used herein, the term “ligation” refers to covalent bond formation or linkage between the termini of two or more nucleic acids (e.g., oligonucleotides or polynucleotides).
As used herein, the phrase “extendable probe terminus” refers to a terminus of a nucleic acid to which another nucleic acid can be ligated.
As used herein, the phrase “non-extendable probe terminus” refers to a terminus of a nucleic acid to which another nucleic acid cannot be ligated without modification. For example, the terminus may be a nucleotide that lacks a 5′ phosphate or a 3′ hydroxyl group. Alternatively, the terminus may be a nucleotide residue with a blocking group attached that prevents ligation.
As used herein, the phrase “universal base” refers to a base that can pair with more than one of the bases typically found in naturally occurring nucleic acids and that can thus substitute for naturally occurring bases in a duplex. The base need not be capable of pairing with each of the naturally occurring bases. Universal bases are described in International Publication No. WO 2006/084132 A2.
As used herein, the phrase “surface functional groups” refers to functional groups that are attached to a surface. These groups can be attached directly to the surface or indirectly to the surface via a linking group.
As used herein, the term “particle” and the phrase “particulate material” are used interchangeably and refer to any solid body having finite mass and internal structure. Exemplary particles include beads and microspheres. According to some embodiments, the particles can have a diameter of less than 100 μm (e.g, 1 μm). Particles can be made of a variety of materials including polymers (e.g., polystyrene), glass and ceramics. Other exemplary particles include magnetic particles. Suitable magnetic particles include, but are not limited to, those disclosed in U.S. Pat. No. 5,512,439. For example, the magnetic particles can be monodisperse superparamagnetic beads produced according to EP 83901406.5 wherein the term “monodisperse” encompasses size dispersions having a diameter standard deviation of less than 5%. The monodisperse particles can have a specific gravity in the range 1.1 to 1.8 or 1.2 to 1.5. The monodisperse particles can be spherical beads having a diameter of at least 1 and not more than 10 microns or at least 2 and not more than 6 microns in diameter (e.g. about 3 microns in diameter).
Sequencing by Oligonucleotide Ligation and Detection (SOLiD™) involves attachment of DNA target to a small, insoluble structure (e.g., a 1 micron diameter cross-linked polystyrene bead) followed by immobilization of a plurality of the structures, where each structure comprises a unique DNA sequence, onto a flat surface. Sequencing techniques of this type are disclosed in International Publication No. WO 2006/084132 A2.
Methods of attachment of the beads to the support have utilized a flat glass microscope slide irreversibly coated with streptavidin DNA laden polystyrene bead with biotinylated nucleotides (e.g., obtained by the action of biotinylated dNTP's and terminal deoxytransferase on the DNA target subsequent to attachment to the bead). Incubation of the biotinylated beads with the streptavidin coated slide results in immobilization of the beads onto the slide by the interaction of streptavidin with the biotin. While kinetically this is a very effective attachment scheme, movement of the beads on the slide was sometimes observed under the conditions required by the DNA sequence assay. When beads are present in high densities on the slide (e.g., up to 100,000 beads/mm2) and interrogated multiple times (e.g., up to 25 times), any significant bead movement can preclude robust identification of a particular bead on subsequent scans within a dense population of beads.
As described herein, a covalent system for bead immobilization has been developed that reduces movement of the beads during sequencing and other forms of genetic analysis. A method is provided that comprises: reacting a nucleophilic group on the surface of a substrate with a molecule comprising a plurality of electrophilic groups thereby providing one or more free electrophilic groups on the surface of the substrate; and reacting nucleophilic groups on a surface of a particulate material with the one or more free electrophilic groups on the surface of the substrate to covalently attach the particulate material to the substrate.
As shown in
As described below, a DNA target nucleic acid that had been covalently attached to a 1 micron cross-linked polystyrene bead was modified by the action of aminoalkyl dNTP's and terminal deoxytransferase on the DNA target subsequent to attachment to the bead. The nucleophilic amino group on the DNA target could then react with the residual electrophilic group of the support surface to form multiple stable covalent bonds between the bead and the glass surface.
Some examples of molecules with multiple electrophilic groups that can be used to bridge the electrophilic surface with the electrophilic bead are shown in
Referring to
When the activated surface containing electrophilic groups is contacted with a particle containing nucleophilic groups, the linkage between the particle and the surface can be characterized as A1-(L-E2-)nL-A3 where at least one of Ax forms a stable covalent linkage to the surface, and at least one of Ax forms a stable covalent linkage to the microstructure. For the case of benzene 1,4-diisothiocyanate shown in
It has been found that stable covalent bonds can be formed between a surface containing electrophilic groups and particles containing nucleophilic groups. In addition, beads containing nucleophilic amino groups from the action of amino-dNTP's and terminal deoxytransferase on a DNA target can be immobilized under aqueous basic conditions on the modified surface. For example, surfaces comprising amino groups that have been activated with benzene 1,4-diisothiocyanate can be used to immobilize beads with nucleophilic groups. In addition, the covalent attachment appears to be quite stable, and no bead movement is observed.
The surface immobilized beads described herein can be used in methods of analyzing nucleic acid sequences based on repeated cycles of duplex extension along a single stranded template via ligation. Sequencing methods of this type are disclosed in U.S. Pat. Nos. 5,750,341; 5,969,119; and 6,306,597 B1 and in International Publication No. WO 2006/084132 A2. Each of these publications is incorporated by reference herein in its entirety. Moreover, the techniques described in the aforementioned publications can be used to analyze (e.g., sequence) nucleic acid templates attached to particles that are bound to supports as described herein. The immobilized beads can be used in sequencing methods that do not necessarily employ a ligation step, such as sequencing using labeled nucleotide that have removable blocking groups that prevent polynucleotide chain extension (e.g., U.S. Pat. Nos. 6,664,079; 6,232,465; and 7,057,026. The immobilized beads can be used in a variety of techniques in which signals on the beads are repeated detected through multiple cycles.
For example, a method is provided that comprises:
(a) hybridizing a first initializing oligonucleotide probe to a target polynucleotide to form a probe-target duplex, wherein the oligonucleotide probe has an extendable probe terminus, wherein the target polynucleotide is attached to a particulate material and wherein the particulate material is covalently attached to the surface of a solid support;
(b) ligating a first end of an extension oligonucleotide probe to the extendable probe terminus thereby forming an extended duplex containing an extended oligonucleotide probe, wherein the extension oligonucleotide probe comprises a cleavage site and a detectable label;
(c) identifying one or more nucleotides in the target polynucleotide by detecting the label attached to the just-ligated extension oligonucleotide probe;
(d) cleaving the just-ligated extension oligonucleotide probe at the cleavage site to generate the extendable probe terminus, wherein cleavage removes a portion of the just-ligated extension oligonucleotide probe that comprises the label from the probe-target duplex; and
(e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the target polynucleotide is determined.
The cleavage site can be cleaved under conditions that will not cleave phosphodiester bonds. Cleavage can therefore occur under conditions that will not cleave the phosphodiester bonds of the extended oligonucleotide probe.
The detectable label can be a fluorescent moiety.
A second end of the extension oligonucleotide probe opposite the first end can comprise a non-extendable probe terminus. This prevents multiple ligations from occurring during a single cycle.
The extension oligonucleotide probe can be an octamer. The oligonucleotide probe can have a sequence as set forth below:
*NNNNN-zzz
wherein each “N” represents, independently, A, C, T or G, “z” represents a universal base, “*” represents the ligation site and “-” represents the cleavage site. The detectable label can be attached to one of the universal bases.
According to some embodiments, four different categories of probes can be used, each having a different label:
*NNNTA-zzz;
*NNNGG-zzz;
*NNNTC-zzz; and
*NNNAT-zzz.
According to some embodiments, the method further comprises hybridizing a second initializing oligonucleotide probe to the target polynucleotide to form a probe-target duplex and conducting steps (b), (c) and (d) repeatedly wherein the second initializing oligonucleotide probe differs by one nucleotide in length from the first initializing oligonucleotide probe. in this manner, the sequence of the target can be determined.
Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
Conjugation of a P1 Oligonucleotide with Carboxylic Acid Polystyrene Beads
The method described below is based on activation of carboxylic groups on the beads surface with 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and in-situ coupling the formed O-acylisourea intermediate with an amino-modified oligonucleotide in the presence of imidazole, creating a covalent bond between the bead surface and the 5′ end of the oligonucleotide. Since bead capacity for the oligonucleotide molecules is limited, there are many free carboxyls left on the bead surface after P1 conjugation. These charged groups interfere with the downstream procedures by causing beads to absorb to fluorescent dyes, thus substantially increasing fluorescent noise. Also, non-capped DNA-loaded beads tend to aggregate. To inactivate these carboxyls, a second procedure, capping these groups with amino-methoxy PEG12, is performed.
The goal of this experiment was to achieve covalent attachment of P1 oligonucleotide on carboxy beads at the same capacity as had been achieved using biotinylated P1 and streptavidin beads. That level of P1 oligonucleotide loading was sufficient for SOLiD™ ligation sequencing. However, using the conjugation system recommended by the bead manufacturer, levels of covalent P1 loading at about 70% of the streptavidin-based system were observed (data not shown). Further experimentation revealed that the presence of both NaCl and DMSO in the covalent system resulted in more efficient P1 conjugation. The effect the ratio of NaCl (“Salt”) and DMSO is illustrated in
As shown in
A typical P1 conjugation procedure is set forth below:
An aliquot of MyOne Carboxylic Acid beads is washed once with 0.01N NaOH, and then five times with DEPC Water. A reaction mixture contains 200 mM NaCl, 0.1 mM 5′-aminomodified P1 oligonucleotide 41 bases long, 1 mM imidazole chloride, 50% v/v DMSO and 200 mM EDC. The beads are mixed well with reagents, vortexed, sonicated and incubated overnight at room temperature on a rotator. The second step (i.e., capping) is done by converting remaining carboxyls into amino-reactive NHS-ester in presence of 200 mM EDC and 50 mM NHS, with subsequent conjugation with aminoPEG12 at 20 mM.
The following describes the development of a novel covalent chemistry involving random deposition of DNA-template beads onto a modified glass surface for the SOLiD™ sequencing platform.
The development of this chemistry involves three major steps. First, optimization of thioureayl formation between amines and isothiocyanate for QC assays and bead immobilization. Second, generation of an electrophilic glass surface by activation of aminopropyl/trialkoxysilane-coated glass with 1,4-phenylenediisothiocyanate (PDITC), Third, addition of nucleophilic amines to the 3′ end of DNA templates by terminal deoxytransferase mediated addition of aminoallyl-dUTP. The development of each step will be discussed with accompanying data, followed by validation of bead immobilization that provides stability under conditions required by the DNA sequence assay.
Proper reaction of amines with isothiocyanate moieties results in thioureayl bond formation, a covalent attachment that is very stable. To determine the optimal conditions for thioureayl bond formation, one of the components in this reaction was fluorescently labeled and used as a reporter. Specifically, these assays used fluorescein-labeled isothiocyanate (FITC). Optimization of this reaction condition not only ensured proper covalent attachment of the bead to the slide but provided quality checkpoints for generating nucleophiles on the bead and electrophilic groups on the slide prior to bead immobilization.
To optimize the reaction conditions, chemistry was performed on beads known to have high amine content on their surface. These beads were reacted with FITC in various buffer types while varying the temperature and pH. Bead fluorescence attributed to the covalent attachment of the fluorescein-labeled isothiocyanate group to amines was measured and compared between different reaction conditions. To ensure covalent bond formation, fluorescence was measured after the same beads were treated with alkali and heavy metal solutions (similar to conditions seen in certain cycles of SOLiD™ sequencing). The abbreviated results from these experiments are shown in
The successful covalent attachment of oligonucleotides to glass supports using PDITC chemistry has been previously reported {Guo et al., Direct Fluorescence Analysis Of Genetic Polymorphisms By Hybridization With Oligonucleotide Arrays On Glass Supports, Nucleic Acids Res. 22(24):5456-65 (1994)}. Experiments were conducted to determine whether these methods could be applied to covalent attachment of 1-micron beads to a modified glass surface. Nucleophilic glass surfaces are commercially available in the form of amine-coated silicate slides that are easily generated by immersing slides in a solution containing (aminopropyl)trialkoxysilanes. These were purchased from various vendors and the relative amine content on each slide was determined by incubating each slide in 200 μM FITC solution, followed by visualization under a fluorescent microscope. After comparing fluorescence on each slide type, it was decided to continue our work using Schott A+ slides, as they reported the highest and most uniform distribution of ITC-mediated fluorescence.
Next, the nucleophilic moieties on the glass surface were converted to electrophiles by reacting the slides overnight in a DMSO solution containing 50 mM PDITC and 20 mM n,n-Diisopropylethylamine (DIEA). DIEA was added as a base to facilitate thioureayl formation between the primary amines and isothiocyanate moiety. Afterward, slides were washed twice with DMSO, then three times with 70% ethanol, followed by three water washes. Slides were then spun dry and stored in an electronic dessicator.
To determine whether activation of the slide surface to electrophiles was successful, slides were incubated with fluorescein-labeled cadaverine, a small and highly reactive diamine. The amount of fluorescence on the slide was measured using a fluorescent microscope. To assess background levels of fluorescence attributed to non-specific binding of the fluorescein, fluorescein-labeled carboxylic acid was included. Additionally, ethanolamine was included in a subset of incubations to see whether it could compete with cadaverine-binding to the slide. As
Another method of determining the reaction progression of PDITC activation involved assessing the amine content of A+ slides before and after incubation with PDITC. Slides were incubated with fluorescein-labeled isothiocyanate (FITC) and visualized on an axon scanner to assess their amine content. Comparisons were made to blank slides, A+ slides, and PDITC-activated slides.
Concurrent with PDITC-activation of the silicate surface, experiments were designed to generate a nucleophilic group on the DNA template beads that would react with the electrophile present on the glass slides. Since terminal deoxytransferase (TdT) had previously been used to add biotin-labeled nucleotides to the 3′ ends of DNA templates on the bead, we decided to continue with this strategy but instead use aminoallyl-labeled dUTP as the addition substrate. The standard recipe that was recommended by the manufacturer of the terminal deoxytransferase was used. The addition of nucleophilic amines by reacting TdT-extended beads with FITC was verified. Streptavidin-coated beads (containing many amines) and carboxy beads were used as positive and negative controls, respectively.
Quantitative analysis of this reaction reveals a dose-dependent increase in the amount of fluorescence as the TdT-extension is allowed to proceed longer as shown in
The Applied Biosystems Sequencing by Oligonucleotide Ligation and Detection (SOLiD™) platform utilizes a plurality of clonal DNA-template beads randomly distributed on a slide surface to which various cycles of biochemistry is applied. For accurate detection and registration of DNA sequences, the beads should remain immobilized for the full duration of the sequencing cycles. To demonstrate that the thioureayl attachments linking the bead to the slide can withstand SOLiD™ sequencing conditions, TdT library beads were deposited on a PDITC-activated slide and bead movement was measured after several modules of SOLiD™ sequencing. Overlay of bead images before and after treatment indicates that beads containing nucleophilic amines are stable on PDITC-activated slides (
While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be appreciated by one skilled in the art from reading this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60935649 | Aug 2007 | US |
