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Surface chemistry and deposition techniques

Surface chemistry and deposition techniques description/claims

This application claims priority to and benefit of U.S. Provisional Application Ser. No. 60/899,173, filed on Feb. 2, 2007, the entire content of which is incorporated herein by reference.

In certain applications, such as single molecule DNA sequencing or the evaluation of polymerases, it is necessary to wash labeled biomolecules across a surface. This process inevitably results in the nonspecific binding of labeled molecules to the surface and a concominant increased background fluorescence and false-positive features. Many surface attachment chemistries have intrinsic properties designed to enhance specific molecule binding but do little to directly inhibit or suppress the effects of the nonspecific binding of fluorescently labeled molecules. The corresponding increase in background fluorescence as labeled molecules are washed across the surface, combined with the limited fluorescent intensity and lifetime of any single fluorophore, imposes restrictions on the overall imaging capabilities of any single molecule surface chemistry. The spatial resolution of an optical system is limited by the Rayleigh criterion: dR=0.61λ/N.A., where λ is the wavelength of collected photons and N.A. is the numerical aperture of the system. As a result of these optical limitations, current methods for the surface deposition and visualization of fluorescently-labeled single molecules suffer from a number of fundamental limitations. Poisson statistics reveal that a certain fraction of all randomly distributed molecules on a surface will be located within a diffraction limit distance of at least one other molecule, resulting in neither of the two molecules being easily resolvable. As a result, for a given optical setup, as the number of deposited molecules increases, the total number of resolvable molecules reaches a maximum and then decreases. Recent a posteriori methods have been developed using centroid localization or photobleaching to resolve multiple single molecules within a diffraction-limited area with high precision. While some of these techniques could conceivably be used to increase the maximum number of resolvable molecules while using random deposition, they would require precise observation of every photobleaching event to realize a significant degree of accuracy. Given the sensitivity and capture rate limitations of current CCD technology, it would likely not be practical to use these methods to completely resolve a highly dense surface array of single molecules.

Common surface attachment chemistries, for both single molecule and bulk sample surface immobilization, typically involve specific ligand binding, specific covalent coupling, or nonspecific chemiabsorption or physiabsorption. Some examples include biotinstreptavidin or digoxygenin-antidigoxygenin coupling, azide-alkyne cycloaddition coupling (24), coupling between an amine-reactive substrate (eg. aromatic isothiocyanate) and a chemically modified aliphatic 1° amine biomolecule, or absorption to a positively charged poly-electrolyte surface followed by chemical or photochemical crosslinking. Many of these surface attachment chemistries have intrinsic properties designed to enhance specific molecule binding but some of them do little to inhibit the effects of nonspecific binding. However, the corresponding increase in background fluorescence as successive fluorescently labeled molecules are washed across the surface limits the overall imaging capabilities of any surface chemistry.

Various other chemical techniques have been developed to minimize background noise while optically imaging single molecules immobilized on a surface. These methods include using fluorescence resonance energy transfer (FRET) to resolve the relative proximity of molecules beyond the diffraction limit, building a negatively charged surface out of a polyelectrolyte multilayer to reduce nonspecific binding of fluorescently labeled nucleotides, using photo-cleavable or chemically cleavable fluorescent labels and extensive washing, and the use of a “smart” hydropolymer shield capable of preventing small molecule binding.

However, there remains a need in the art for a method of resolving single molecules on a surface, especially when random deposition of the molecules is desired.

The present invention provides surface chemistry for the visualization of labeled single molecules (analytes) with improved signal-to-noise properties. According to one aspect of the invention, analyte molecules to be observed are bound to surface attachment features that are spaced apart on the surface such that when the analytes are labeled adjacent analytes are optically resolvable from each other. One way to express this concept is that binding elements should be spaced apart such that the Guassian point spread functions of adjacent labels do not overlap. Another way of expressing this concept is that the surface binding elements should be spaced apart by a distance equal to at least the diffraction limit for an optical label attached to the bound analytes. For purposes of the invention, an analyte is any molecule that one wishes to observe. Preferred molecules are nucleic acids, proteins and other biomolecules.

The precise spacing of binding elements depends upon the label used. Diffraction limits of various optically-detectable labels are well known and can be selected at the convenience of the user. In one embodiment, a low-autofluorescence glass (e.g., a coverslip) is coated with a thin metal film and a specific surface coupling chemistry is applied for the attachment of labeled molecules. The metal film may be any appropriate metal (examples are provided below) at the convenience of the user and is applied such that total internal reflection illumination can be conducted on the surface. An evanescent field is generated by total internal reflection and is enhanced by the production of surface plasmons from the thin metal film, which increases the intensity of fluorescently labeled molecules within approximately 150 mn of the surface. This is explained by the fact that surface plasmons tend to stay longer along the surface than the evanescent field, and the electromagnetic field produced by the surface plasmons is intensified near the metal surface. The presence of the thin metal film quenches excited fluorophores near the surface (within tens of mn) by a mechanism of fluorescent energy transfer into the surface plasmon modes of the metal.

The invention also provides surface deposition methods that are useful independent of the surface chemistry being used. These deposition strategies allow for the fabrication of fully-resolved single molecule arrays at a feature density that surpasses that of any previously-described methods. Methods and devices of the invention improve the total resolvable molecule limit by selectively spacing deposited molecules at least a diffraction limit apart from every other molecule.

A particular application of methods and materials of the invention is for single molecule sequencing of nucleic acids. In particular, methods of the invention allow increased resolution of ordered arrays of nucleic acid duplex on surfaces for sequencing. In a preferred embodiment, primers for nucleic acid synthesis are placed on a surface as described herein. For ease of use, the primers can be universal primers, such as poly-T sequences. Genomic DNA (or RNA or cDNA copies of RNA) are then sheared and, in the case in which primers are poly-T, tailed with a polyadenylation sequence in order to hybridize to the primer (e.g., with a terminal transferase enzyme as is well-known in the art). After appropriate wash steps, labeled nucleotides are added in the presence of a polymerase enzyme for template-directed sequencing-by-synthesis. The resulting sequencing reactions allow visual observation of individual incorporated nucleotides in sequence. A general approach to single molecule sequencing is described in co-owned, U.S. Pat. No. 7,282,337, incorporated by reference herein.

Further aspects and features of the invention are provided below in the detailed description thereof.

FIG. 1 shows a sample DNA construct for diffraction limit spacing surface attachment. R=Type IIs restriction enzyme recognition site; c=Type IIs restriction enzyme cut site. The fluorophore may alternatively be attached to the 3′ end of the lower strand. Following cleavage, the dsDNA in lowercase letters is removed and washed away.

FIG. 2 shows the results of a computer simulation showing the maximum number of resolvable molecules as a function of the diffraction limit and the physical size of the photon detector for a variety of different single molecule deposition methods. ‘O’=random deposition method; ‘+’=random deposition method with Selvin resolution of single molecules; ‘x’=bin deposition (see Variations); ‘v’=bead deposition. Relative pixel size=(detector length/dR)2

FIG. 3 shows a variant scheme for single molecule diffraction limit spacing with a universal probe annealing mechanism. Single molecules are deposited on the surface as before with a cleavable linker on the bead. Following deposition, the bead is cleaved off and a poly-T modified single stranded DNA, is annealed. Sequencing by synthesis of the unknown template may then readily proceed.

• Provisional Patent
• Utility Patent

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