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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • B01J2219/00603—Making arrays on substantially continuous surfaces
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  • B01J2219/00677—Ex-situ synthesis followed by deposition on the substrate
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  • 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
  • C12Q1/6841—In situ hybridisation

Definitions

• the invention relates generally to alignment and controlled spacing of nucleic acid molecules on a substrate for biological analysis.
• Methods exist to perform low-density molecular alignment of multi-stranded nucleic acid molecules in a thin or monolayer on a substrate. Some focus on isolating one or a few strands of materials and stretching them out for observation and genetic analysis. Examples of such methods are molecular combing using an air-water meniscus in US Patent 6,548,255 and a molecular alignment technique for optical mapping as described in US Patent 6,147,198 and US Patent Application 2005/0082204. However, a need exists for new methods to perform high-density molecular alignment of multi-stranded nucleic acid molecules.
• the invention provides methods to perform high-density molecular alignment of nucleic acid molecules.
• the invention provides a high density spacing nucleic acid molecule monolayer.
• the nucleic acid molecules are multi-stranded.
• the present invention includes methods of performing molecular alignment of multi-stranded nucleic acid molecules on a thin-membrane substrate which results in a monolayer or partial monolayer of molecules having significantly less space between them than other methods.
• the first step of the methods includes attaching one end of the molecules to the substrate surface in a predetermined pattern or density.
• the second step of the method includes applying one or a combination of alignment forces (simultaneously or in series) to the molecules so that they straighten-out and align in the desired direction.
• the predetermined attachment pattern and use of spatial density techniques results in the molecules being closely spaced after application of the alignment forces, such as fluid flow.
• the aligned molecules will bind to the substrate surface.
• the substrate surface is modified to allow for binding of the aligned molecules.
• a third step includes fixing the aligned molecules to the surface to maintain the high-density alignment pattern. These steps may be repeated on the same substrate to create more complex patterns.
• the invention provides a method of generating a high density spacing nucleic acid monolayer, the method comprising performing a first attachment step to attach one end of nucleic acid molecules of a first population of nucleic acid molecules to a substrate at a first group of contact points, wherein the first group of contact points are located in an attachment area on a surface of a substrate, and aligning the nucleic acid molecules attached to the first group of contact points to produce a first set of aligned nucleic acid molecules that are substantially parallel and are spaced less than 1 micron apart, whereby the first set of aligned nucleic acid molecules bind to the surface of the substrate, thereby generating a high density spacing nucleic acid molecule monolayer on the surface of the substrate that comprises the first set of aligned nucleic acid molecules.
• the method further comprises performing a second attachment step to attach a second population of nucleic acid molecules to the attachment area at a second group of contact points, and aligning the nucleic acid molecules attached to the second group of contact points to produce a second set of aligned nucleic acid molecules that are substantially parallel and are spaced less than 1 micron apart, whereby the second set of aligned nucleic acid molecules bind to the surface of the substrate, thereby generating a high density spacing nucleic acid molecule monolayer on the surface of the substrate that comprises the first and second set of aligned nucleic acid molecules.
• the first attachment comprises the attachment of the nucleic acid molecules of the first population to the first group of contact points through a first group of linkers.
• the second attachment comprises the attachment of the nucleic acid molecules of the second population to the second group of contact points through a second group of linkers.
• the first linker is an oligonucleotide.
• the second linker is an oligonucleotide.
• the first linker comprises an n-octyl group.
• the second linker comprises an n-octyl group.
• the first or second attachment step comprises attaching the nucleic acid molecules by forming one or more covalent bonds between the surface of the substrate and the nucleic acid molecules.
• the first or second attachment step comprises UV-crosslinking, producing ionic attraction between phosphate groups and a functionalized surface, using modified DNA to bond to other substrate surface modifications and/or using cationic surface treatment.
• the aligning comprises directing a fluid flow across the substrate. In some embodiments the aligning comprises applying an electric field to the nucleic acid molecules. In some embodiments the aligning comprises applying a magnetic field to the nucleic acid molecules.
• the substrate comprises an alignment area.
• the aligning causes the nucleic acid molecules to be aligned on the alignment area of the substrate.
• the substrate comprises a thin membrane.
• the thin membrane is a nanometer scale membrane.
• the aligned nucleic acid molecules are substantially straight.
• the substrate comprises a graphite surface.
• the aligned molecules are in a ladder conformation.
• the nucleic acid molecules of the first population are labeled nucleic acid molecules. In some embodiments the nucleic acid molecules of the first population are unlabeled nucleic acid molecules. In some embodiments the nucleic acid molecules of the second population are labeled nucleic acid molecules. In some embodiments the nucleic acid molecules of the second population are unlabeled nucleic acid molecules. In some embodiments the labels are detectable using a transmission electron microscope (TEM). In some embodiments the labels comprise atom(s) having a nuclear charge that is detectable in contrast to background noise on an image from a TEM. In some embodiments the labels comprise one to five non-fluorescent atoms.
• TEM transmission electron microscope
• the nucleic acid molecules are at least 100 base pairs in length. In some embodiments the nucleic acid molecules are at least 1000 base pairs in length. In some embodiments the nucleic acid molecules are at least 10,000 base pairs in length. In some embodiments the nucleic acid molecules are at least 20,000 base pairs in length.
• binding of the aligned molecules to the surface of the substrate is non-covalent. In some embodiments the binding of the aligned molecules to the surface of the substrate is reversible. In some embodiments binding to the surface comprises slowing and/or stopping directional fluid flow, changing temperature of fluid and/or environment, drying the nucleic acid molecules in a directional fashion after fluid flow is stopped, and/or adding reagents and/or activators that promote binding to the surface before or after fluid is removed. In some embodiments binding to the surface comprises attaching the nucleic acid molecules by forming one or more covalent bonds between the surface of the substrate and the nucleic acid molecules.
• binding to the surface comprises UV-crosslinking, producing ionic attraction between phosphate groups and a functionalized surface, using modified DNA to bond to other substrate surface modifications and or using cationic surface treatment.
• linkers and surface modifications are in the same area on the substrate.
• binding to the surface comprises attaching the nucleic acid molecules to the surface through a binding pair.
• at least a part of the substrate . surface is hydrophobic.
• at least a part of the substrate surface is functionalized to be hydrophobic.
• the high density spacing nucleic acid molecule monolayer is a partial monolayer.
• At least 25% of the aligned nucleic acid molecules are spaced less than 100 nm apart. In some embodiments at least 50% of the aligned nucleic acid molecules are spaced less than 100 nm apart. In some embodiments at least 25% of the aligned nucleic acid molecules are spaced less than 10 nm apart. In some embodiments at least 50% of the aligned nucleic acid molecules are spaced less than 10 nm apart. In some embodiments at least 25% of the aligned nucleic acid molecules are touching. In some embodiments at least 50% of the aligned nucleic acid molecules are touching.
• At least 25% of the aligned nucleic acid molecules are aligned as alternating labeled and unlabeled nucleic acid molecules. In some embodiments at least 50% of the aligned nucleic acid molecules are aligned as alternating labeled and unlabeled nucleic acid molecules.
• one or more magnetic beads are attached to the nucleic acid molecules and the nucleic acid molecules are aligned by applying a magnetic force.
• the invention provides a high density spacing nucleic acid molecule monolayer comprising a substrate, and aligned nucleic acid molecules spaced less than 1 micron apart attached to the substrate.
• the monolayer comprises a first population of nucleic acid molecules attached to the substrate at a first group of contact points. In some embodiments the first group of contact points are located in an attachment area on the surface of the substrate. In some embodiments the monolayer comprises a first set of aligned nucleic acid molecules attached to the surface of the substrate at a plurality of points. In some embodiments the monolayer comprises a second population of nucleic acid molecules attached to the substrate at a second group of contact points. In some embodiments the second group of contact points are located in the attachment area on the surface of the substrate.
• the substrate has an alignment area. In some embodiments the substrate has a graphite surface. In some embodiments of the monolayers described herein the aligned nucleic acid molecules comprises a second set of aligned nucleic acid molecules attached to the surface of the substrate at a plurality of points. In some embodiments the aligned nucleic acid molecules are substantially straight. In some embodiments the aligned nucleic acid molecules are in ladder conformation. In some embodiments the aligned nucleic acid molecules are labeled nucleic acid molecules.
• the nucleic acid molecules are a mixture of labeled and unlabeled nucleic acid molecules.
• the labels are detectable using a transmission electron microscope (TEM).
• the labels comprise atom(s) having a molecular weight that is detectable in contrast to the background noise on an image from a TEM.
• the labels comprise one to five non-fluorescent atoms.
• the nucleic acid molecules are at least 100 base pairs in length. In some embodiments the nucleic acid molecules are at least 1000 base pairs in length. In some embodiments the nucleic acid molecules are at least 10,000 base pairs in length. In some embodiments the nucleic acid molecules are at least 20,000 base pairs in length. In some embodiments the nucleic acid molecules are attached to the surface of the substrate by linkers. In some embodiments the nucleic acid molecules are attached to the surface of the substrate by one or more covalent bonds between the surface of the substrate and the nucleic acid molecules.
• the substrate surface is hydrophobic.
• the substrate has a thin membrane.
• the thin membrane is a nanometer scale membrane
• at least a part of the substrate surface is functionalized to be hydrophobic.
• the monolayer is a partial monolayer.
• At least 25% of the aligned nucleic acid molecules are spaced less than 100 nm apart. In some embodiments at least 50% of the aligned nucleic acid molecules are spaced less than 100 nm apart. In some embodiments at least 25% of the aligned nucleic acid molecules are spaced less than 10 nm apart. In some embodiments at least 50% of the aligned nucleic acid molecules are spaced less than 10 nm apart. In some embodiments at least 25% of the aligned nucleic acid molecules are touching. In some embodiments at least 50% of the aligned nucleic acid molecules are touching.
• the aligned nucleic acid molecules are aligned as alternating labeled and unlabeled nucleic acid molecules. In some embodiments at least 50% of the aligned nucleic acid molecules are aligned as alternating labeled and unlabeled nucleic acid molecules. In some embodiments of the monolayers described herein the monolayers are produced by any of the methods described herein.
• the invention provides a method for determining the sequence of a population of nucleic acids comprising obtaining a population of labeled nucleic acid molecules, generating a high density spacing nucleic acid molecule monolayer using the population of labeled nucleic acid molecules as the first or the second population of nucleic acid molecules according to any of the methods described herein, and reading the sequence of the nucleic acid molecules of the monolayer using a particle beam.
• the method further comprises generating the population of labeled nucleic acid molecules by obtaining a population of nucleic acid molecules from a sample, and labeling nucleotides of the nucleic acid molecules with one or more labels that are detectable using a transmission electron microscope (TEM) to generate the population of labeled nucleic acid molecules.
• TEM transmission electron microscope
• FIG. 1 shows an overview of the method steps.
• FIG. 2 shows an overview of the alignment process.
• FIG. 3 shows an overview of the alignment process using a larger attachment area.
• FIG. 4 shows a substrate attachment area.
• FIG. 5 shows the use of labeled and unlabeled DNA in the process.
• FIG. 6 shows overlapped fluorescence micrographs (scale given by window; white line shows width ⁇ 60 ⁇ m) showing elongated lambda DNA molecules deposited on the flat portions of the surface. DNA molecules are stained with YOYO-I.
• FIG. 7 shows different location on the same substrate as shown in Figure 6, but with an adjusted plane of focus. Note that some molecules deposited on the window are now in focus.
• FIG. 8 shows two images of the same location on a substrate.
• the left panel shows the outline of the window structure imaged by fluorescence microscopy bearing arrayed, but out-of-focus lambda DNA molecules.
• the right panel shows how altering the plane-of-focus allows sharp imaging of molecules at the crest of the distorted window.
• FIG. 9 shows an image taken of a substrate; different locations show the deposition of lambda DNA molecules.
• the window distortion is made apparent by the presence of out-of- focus DNA molecules.
• FIGS. 10 and 1 1 show TEM images of a substrate with DNA molecules with a pitch of 200 nm prepared using the methods of the invention
• FIG. 12 shows an embodiment of the invention in which the molecules are aligned in the attachment area.
• FIG. 13 shows an embodiment of the invention in which pilot molecules are used to create a spacing for the analyte molecules (the nucleic acid molecule to be analyzed).
• FIG. 14 shows a TEM image of ladder-conformation DNA. The double-stranded
• Each of the parallel lines is one of the two double-strands.
• the spacings between the dark lines alternate between 1.5 nm and 2 nm.
• the 2 nm distances are distances between the labels of two strands of the same molecule.
• the 1.5 nm distances are the spacings between labels of adjacent strands of adjacent molecules.
• the overall molecular pitch is about 3.5 nm.
• An "Attachment Area” is a defined surface area on a substrate where molecules are allowed or caused to bind to the substrate surface by covalent binding or other technique(s) known to those of ordinary skill in the art.
• the attachment area is the area where the contact points are located for the attachment of the linkers and/or nucleic acid molecules. In some embodiments the attachment area is also the area where the nucleic acid molecules align and where the high density spacing monolayer is formed.
• a substrate may have one or more attachment areas.
• An “Alignment Area” is defined as the area of the substrate where the nucleic acid molecules align and form the high density spacing monolayer.
• the alignment area is located adjacent to the attachment area.
• the alignment area may have surface modifications to allow for the binding of the aligned molecules to the alignment area.
• a substrate may have one or more alignment areas.
• a “Thin Membrane” is an area of a substrate that has a smaller thickness.
• the thin membrane can be less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, less than 10 nm, less than 8 nm, less than 5 nm, and less than 3 nm. In some embodiments the thin membrane is less than 30 nm. This thickness is well suited for TEM (Transmission Electron Microscope) analysis of the aligned molecules.
• TEM Transmission Electron Microscope
• the thin membranes areas have a combination of thinness and material that is sufficiently transparent to particle beam species of a particle beam to allow detection of interactions of the particle beam species with a biological sample on the surface of the one or more transparent areas after the particle beam species passes through the sample and the transparent area.
• the thin membrane can be part of the alignment area or part of the attachment area. In some embodiments the thin membrane is a nanometer scale membrane.
• Contact points as defined herein, are specific points on a substrate, optionally on an attachment area of the substrate, to which the linkers or the nucleic acid molecules can be attached.
• High Density Spacing after molecular alignment is defined as spacing between molecules that, after alignment, is less than 1 micrometer, less than 500 nanometers (nm), less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 25 nm, less than 20 nm, less than 15 nm, less than 10 nm, less than 9 nm, less than 8 nm, less than 7 nm, less than 6 nm, less than 5 nm, less than 4 nm, less than 3 nm, less than 2 nm, less than 1 nm, or 0 nm.
• the molecules are touching each other. Touching is defined as molecules being as close to each other as allowed under a specific set of conditions, including taking into account the geometry of the molecules and repelling interactions between the molecules.
• the spacing is within a range of: a) zero space between molecules on their width (i.e., the molecules are in contact), and b) the space between molecules on their width is ten (10) times the average width of adjacent molecules. However, in all embodiments the spacing will be less than 1 micrometer.
• Length is defined as the dimension of the molecules parallel to the ribose-phosphate backbones. Width is defined as the dimension at right angles to the ribose-phosphate backbone and in the plane of the local substrate surface.
• the average width of the molecules is approximately twenty two (22) Angstroms
• high density spacing would be any spacing on the width dimension in the range of zero (0) Angstroms to two hundred twenty (220) Angstroms.
• the invention also embraces other width / length ratios, as long as the spacing is less than 1 micrometer.
• the molecules may be any length or width, may be made of different materials, may have different dimensions or any combination thereof.
• Linkers include, without limitation, sequence dependent single strand oligomers and non-sequence dependent molecules.
• the linkers are nucleic acids. Other forms of linkers are known to those of ordinary skill in the art.
• the binding between the linker and a DNA molecule may be reversible or non-reversible, covalent or non- covalent.
• the linkers may include one or multiple types of molecules and may be of any length.
• a first linker is used for a first group of nucleic acids while a second linker is used for a second group of nucleic acids.
• the linker is an oligonucleotide that is complementary to the single strand overhang of the nucleic acid molecule.
• the oligonucleotide binds a double stranded DNA sequence.
• the linker is a hydrophobic molecule.
• the linker comprises a medium chain hydrocarbon group such as an n-octyl group. The medium chain hydrocarbon group is capable of binding dsDNA at the appropriate pH (See e.g., Allemand, J.-F., Bensimon, D., et al. 1997. pH-Dependent Specific Binding and Combing of DNA. Biophysical Journal. 2064-2070).
• "Molecular aligned" nucleic acid molecules on a substrate are defined as two (2) or more nucleic acid molecules on a substrate in substantially parallel or parallel lines.
• High quality alignment has no overlap of the nucleic acid molecules at any point, with nucleic acid molecules in locally parallel lines, with nucleic acid molecules being substantially straight along their entire length.
• Acceptable quality molecular alignment can include overlaps of molecules and substantially parallel but curved lines, or partial regions of tangles within a generally straightened and aligned molecule.
• Nucleic acid molecules include, but are not limited to, deoxyribonucleic acid (“DNA”), ribonucleic acid (“RNA”), peptide nucleic acid (“PNA”), other nucleic acid polymer analogues and modifications, and mixtures thereof. Nucleic acid molecules can be single stranded or multiple stranded, or a mixture of single stranded and multiple stranded (e.g., having a portion that is single stranded and a portion that is double stranded).
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• PNA peptide nucleic acid
• Nucleic acid molecules can be single stranded or multiple stranded, or a mixture of single stranded and multiple stranded (e.g., having a portion that is single stranded and a portion that is double stranded).
• Multi- stranded nucleic acid molecules include, but are not limited to, double stranded DNA, triple stranded DNA, double stranded RNA, chimeric DNA/RNA double strands, DNA/PNA double strands and RNA/PNA double strands. Additional aspects of nucleic acids are described in international patent publication WO2007/076132.
• a “ladder conformation”, as defined herein, is a conformation in which the two ribose-phosphate backbones have been untwisted from the DNA helix and have become straight and parallel like the two long sides of a ladder that are connected by steps, with the Watson-Crick base pairs forming the steps.
• a ladder conformation can be formed by controlled stretching of the helix away from one attachment point by certain methods of molecular alignment. The ladder form can be maintained by binding the DNA molecule to a surface. The organization of the DNA bases in the ladder conformation allows for improved imaging of nucleic acid base pairs.
• a monolayer is a layer of molecules covering a substrate that has a thickness of one molecule. It should be appreciated that in the invention a "monolayer" refers to both a substrate-covering monolayer and a "partial monolayer".
• a partial monolayer as used herein is a layer of molecules covering a local area of a substrate that has a thickness of one (1) molecule for a substantial portion of the local area and no molecules in other portions. In some embodiments, a portion of the local area can have a thickness of more than one (1) molecule. In addition to covering a local area of a substrate, a partial monolayer may also cover a local area of a thin layer of other molecules.
• a substrate is a material having a rigid or semi-rigid surface.
• At least one surface of the substrate will be substantially flat, although in some embodiments it may be desirable to have flow channels and/or other mechanical alignment aids, such as topological features on the substrate, to facilitate molecular alignment.
• flow channels and/or other mechanical alignment aids include, without limitation, trenches in the surface in which one or more molecules can fit partially or in full and raised barriers between which one or more molecules can fit partially or in full.
• the range of dimensions and materials of a substrate are known to those of ordinary skill in the art.
• the substrates have an attachment area and an alignment area. In some embodiments the substrates do not have an alignment area. In some embodiments the attachment area and alignment area contain different surface functionalizations. In some embodiments the substrates have one or more thin membranes and structural support for the thin membranes.
• the methods of the invention in some embodiments have three basic steps: a step of attaching an end of nucleic acid molecules to a substrate, a step of aligning the nucleic acid molecules, and a step of binding the aligned nucleic acid molecules to the substrate at one or more places along the molecules (except for the previously attached end).
• the last step can be "passive” (mostly through non-covalent binding) or active (performing a reaction to attach the aligned molecules to the substrate).
• the methods of the invention can be used to perform molecular alignment of nucleic acid molecules with high density spacing on a substrate. In some embodiments, the methods are used for performing molecular alignment in a partial monolayer with high density spacing of dsDNA molecules for genetic analysis.
• the DNA molecules have been labeled to improve the quality of genetic analysis.
• Genetic analysis includes sequence determination of the dsDNA molecules and determination of expressed sequences. Genetic analysis also includes the quantitative determination of dsDNA and the determination of differently expressed nucleic acids in a sample. DNA and nucleic acids which are to be genetically analyzed are referred to herein as "analyte DNA”, “sample DNA”, “DNA to be analyzed”, “analyte nucleic acids”, “sample nucleic acid”, and “nucleic acids to be analyzed”.
• the methods are used for performing molecular alignment in a partial monolayer with high density spacing of dsDNA in which some of the nucleic acid molecules have labels that are atom(s) having an nuclear charge (as indicated by Atomic Number, or "Z") that is detectable in contrast to the background noise on an image from a Transmission Electron Microscope ("TEM”), and some of the nucleic acid molecules have no such labels.
• the labeled and/or unlabeled DNA molecules include modifications to support alignment and spacing.
• the method may be used to perform high density molecular alignment of multi-stranded nucleic acid molecules with length greater than, but not limited to: 100, 200, 300, 500, 700, 1000, 1500, 2000, 3000, 5000, 7000, 10000, 15000, 20000, 25000, 30000, 50000, 70000, 100000, 200000, 5000000, 1000000 or more bases.
• Certain embodiments include the use of thin membranes upon which the molecular alignment and spacing is performed followed by TEM analysis.
• the methods are used to determine the sequence of nucleic acid molecules, e.g., dsDNA, in contiguous reads of 5,000 or more, or 10,000 or more, or 20,000 or more, or 50,000 or more, or 100,000 or more, or 500,00 or more, or 1,000,000 or more, base-pairs.
• Figure 1 is a schematic overview of the three steps in the method to be described.
• the first step of the methods of the invention is to complete attachment of one end of a plurality of DNA molecules to a substrate in a controlled pattern.
• the pattern of attachment is chosen to result in a High Density Spacing measurement of DNA molecules that, after alignment, provides or approximates a desired or target spacing suitable for application in genetic analyses.
• the contact points to which the linkers can be attached are dispersed all over the attachment area, and the DNA molecules can be attached to the whole substrate.
• the substrate has both an attachment and an alignment area and the DNA molecules can only be attached to a subset of the substrate area.
• the controlled pattern of attachment of one end of a plurality of DNA molecules can be achieved by a variety of methods.
• the attachment area is equipped with linkers at a predetermined density or pattern. It should be appreciated that the density of the spacing of the linkers does not need to be the same as the final density of High Density Spacing of the aligned DNA molecules, as the spacing density can be regulated by the flow direction during the alignment step (See below).
• the substrates of the invention have a attachment area with a predetermined linker density, and a resulting alignment area where the DNA molecules have a high density spacing.
• Factors that alone or in combination can determine the High Density Spacing of the aligned DNA molecules include: 1) the linker surface density; 2) the attachment area size and shape; 3) the direction of the subsequent molecular alignment technique. Examples of how these factors interact are provided in paragraphs and Figures 1-5. In all embodiments the above factors are arranged to result in a High Density Spacing of aligned molecules.
• the DNA molecules are bound to the linkers in preparation for the alignment step.
• Methods to attach linkers to substrates are known in art, for instance in the creation of DNA microarrays.
• Methods to bind the DNA molecules to the linkers are also known to those of ordinary skill in the art and include, without limitation, a direct covalent bond, hybridization to sequence dependent linkers or other bonds complementary to the type of linker used.
• the linkers are oligonucleotides.
• the length of the DNA molecules to be attached can vary widely and is only limited by the size of the substrate surface area on which the alignment takes place.
• the linker and the DNA molecule are already bound in liquid before diffusion and attachment of the linker to the substrate surface. In some embodiments there are one or more intermediary molecules between the linker and the DNA. In some embodiments the DNA molecules bind directly to the substrate contact points without a separate linker. The DNA molecules previously described may also be single strands that are turned into multiple strands by PCR or another method after attachment to the substrate at one end. In some embodiments non-functional linkers need to be removed to optimize binding of the aligned molecules to the substrate surface as unreacted linker can interact with the alignment of the nucleic acid molecules. The amount of unreacted linker can be minimized by increasing the ratio of nucleic acid molecules to linkers when performing the attachment or by quenching or removing the unreacted linkers.
• the DNA molecules may be labeled or not labeled.
• Types of labels include, but are not limited to, fluorescent molecules, one to five non-fluorescent atoms, including atoms with a high nuclear charge, and radioactive molecules. Additional aspects of nucleic acid labels are described in international patent publication WO2007/076132 and WO2006/019903.
• the molecules that are to be attached to the surface are mixed with other molecules of comparable size, so called “Filler Molecules” or “Pilot Molecules”.
• the pilot molecules are larger in size than the DNA molecules to be analyzed. This mix of molecules preferably is contained in a fluid or other medium to facilitate mixing. These pilot molecules take up a predetermined amount of space on the surface, based upon their proportion in the mix of nucleic acid molecules, therefore diluting the nucleic acid molecules intended for attachment.
• high density spacings comprising both the DNA molecules to be analyzed and pilot molecules can also be prepared by first attaching the pilot molecule to the linkers and in a subsequent step attaching the DNA molecules to be analyzed to the linkers.
• the contacts for the first molecule e.g., a pilot molecule
• the second molecule e.g., a DNA molecule to be analyzed.
• linkers for a first molecule and linkers for a second molecule are arrayed in a predetermined pattern to create an alignment with a predetermined pattern.
• the molecules to be analyzed are attached prior to the attachment of the pilot molecules.
• the pilot molecules and molecules to be analyzed are attached to different kind of linkers (e.g., oligonucleotides with a different sequence).
• the pilot molecules are aligned creating alignment channels into which the molecules to be analyzed can be aligned
• the predeterminedly arrayed linkers or other molecules serve as attachment points for the DNA molecules to be analyzed (Also referred to as analyte molecules).
• the pilot molecules also can incorporate calibration aids, such as periodic labels or specific sequences that indicate e.g., by inference, length or other properties of the nucleic acid molecules or associated labels from the sample.
• the linkers are attached to contact points with a predetermined pattern on the attachment area using techniques including, but not limited to, atomic force microscopy or nano-scale lithographic procedures.
• the linkers are limited to the attachment area by covering the alignment area when adding the linkers to the attachment area, or by using different surfaces for the attachment area and the alignment area, with the alignment area being unreactive towards the linkers.
• the first example describes how the direction of alignment interacts with the attachment area size and shape to affect the High Density Spacing.
• Figures 2A and 2B illustrate this example and show how the direction of the molecular alignment influences the High Density Spacing for a given (non-symmetrical) attachment area.
• the attachment area is a three (3) nanometer ("nm") by seven (7) nm rectangle and the linker surface density results in attachment in the attachment area of the surface of three (3) linkers, which are one (1) nm in diameter.
• linkers are confined to the Attachment Area, stochastic surface binding will result in the linkers forming an approximate line with three (3) molecules spaced one (1) nm apart in the seven (7) nm dimension and, in the three (3) nm dimension, a one (1) nm space between each molecule and the sides.
• Long DNA strands (for instance one thousand (1,000) nm in length) are then attached to the linkers. If the subsequent alignment goes out (i.e., flow is in the direction of) a seven (7) nm side of the rectangular attachment area (in either direction), the High Density Spacing will be approximately one (1) nm.
• the High Density Spacing will usually be zero (0) nm as the upstream DNA molecules (the direction from which the alignment fluid is flowing) will go to the side of the downstream molecules and stay next to each other.
• the second example is shown in Figure 3, which illustrates how the size and nonsymmetrical shape of the attachment area interacts with the direction of the molecular alignment to influence the High Density Spacing.
• the surface density of linker molecules is the same as in the first example, but the Attachment area shape is six (6) nm by seven (7) nm.
• the linkers will diffuse into approximately two (2) lines of three (3) molecules (if confined to the attachment area), like two (2) of the attachment areas in the first example side by side. DNA strands one thousand (1,000) nm in length are then attached to the linkers.
• the High Density Spacing will be approximately zero (0) nm between perhaps four (4) molecules and perhaps all six (6) because there are now upstream DNA molecules to fill-in the spaces between DNA molecules as spaced in the first example.
• the third example shows how different densities of the alignment molecules in a liquid interact with the attachment area size and shape to affect the High Density Spacing.
• Figures 4A and 4B illustrate this effect.
• This example uses one surface density of linkers that is consistent over a whole substrate. Within the three (3) nm by three (3) nm attachment area there are nine (9) linkers. If the surface density of linkers is tripled, then there will be approximately twenty seven (27) molecules in the same size attachment area. This will increase the High Density Spacing after alignment because there will be three (3) times as many upstream DNA molecules (after bonding to linkers) for the same linear width into which the DNA molecules will move to fit during directional alignment.
• the alignment of the nucleic acid molecules can be controlled and selected such that the nucleic acid molecules are less than 1 micron apart. This proximity is surprisingly achieved using the methods described herein, such that unexpectedly close packing of the aligned nucleic acid molecules can be achieved.
• At least 25% of the aligned nucleic acid molecules in the monolayer are spaced less than 1 micron apart, preferably less than about: 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3nm, 2 nm, 1 nm, 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm, 0.3 nm, 0.2 nm, or 0.1 nm apart.
• the molecules are between 2.5nm and 3.5 nm apart.
• the aligned nucleic acids are adjacent.
• the aligned nucleic acids are touching over at least about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of their lengths.
• at least about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the aligned nucleic acid molecules are spaced less than the aforementioned distances apart (or touching each other).
• labeled nucleic acids are aligned in the presence of unlabeled nucleic acids, e.g., pilot molecules.
• the aligned nucleic acid molecules preferably are aligned as alternating labeled and unlabeled nucleic acid molecules, e.g., as shown in Figure 3 and Figure 5.
• the labeled DNA molecules are typically, but not necessarily, the ones from a patient or target sample to be analyzed.
• the labels of the labeled DNA molecules can be of any type known to those of ordinary skill in the art including, but not limited by fluorescent labels and radioactive labels. Labels may also consist of one to five atoms that are significantly heavier than the average DNA molecular weight as further described in published patent applications US Patent 7,291,468, US Patent 7,291 ,467, US Patent 7,288,379, and WO2006/019903, all of which are included herein by reference for the relevant teaching of labels.
• the pilot molecules are unlabeled DNA molecules that preferably are not from the patient or target sample but should have similar physical properties (length, width, density and/or other properties) to the labeled DNA molecules.
• the Pilot Molecules may or may not have the ability to bind to the labeled molecules, e.g., by sequence specific or non sequence specific binding.
• the use of unlabeled nucleic acid molecules provides another way to control the High
• Unlabeled nucleic acid molecules will mix randomly with the labeled nucleic acid molecules. As the percent of unlabeled nucleic acid molecules in the mix is increased, the labeled nucleic acid molecules will be increasingly separated on average (once attached to the surface) and the probability that two labeled nucleic acid molecules will be adjacent and/or touching is decreased. This control is valuable because it may be preferred to have a minimum separation of labeled nucleic acid molecules to increase the effectiveness of the reading systems for the labels. For example, if two labeled molecules are next to each other, the labels of one nucleic acid molecule may be difficult to distinguish or indistinguishable from the labels on the other nucleic acid molecule.
• the natural property of multi-stranded nucleic acid molecules to self-organize will create greater uniformity in molecular alignment when the nucleic acid molecules are right next to one another.
• separation of labeled nucleic acid molecules can still be achieved even when the density of attachment points on the surface are targeted to almost zero High Density Spacing and therefore attain high uniformity.
• sample nucleic acid molecules will usually be labeled before mixing with unlabeled nucleic acid molecules, labeling may be done at any point in the process for this method.
• the pilot molecules are attached and aligned within a substrate area in a first cycle of the method of the invention. Subsequently, a second cycle is performed to attach and align analyte molecules around the pilot molecules in the same substrate area.
• the pilot molecules may be quite long, for example hundreds of thousands of base pairs in a DNA double strand, to maximize the effectiveness and uniformity of the molecular alignment step.
• the pilot molecules are attached along their full length in a manner that will prevent them from detaching during the second cycle with analyte molecules and alignment forces. Generally, the pilot molecules are not labeled to avoid being confused with labeled analyte molecules during post-alignment analysis.
• the pilot molecules may be equipped with labels different form the labels of the analyte molecules to allow for the localization of the pilot and thereby the analyte molecules.
• the pilot molecules can serve as guides or channel edges on the surface of the substrate to improve the alignment of the analyte molecules (See also Fig. 13).
• attachment can occur either on the membrane area or nearby, whereas alignment and spacing control should be substantially on the membrane area of the substrate.
• the attachment area can include both a thin membrane area and another adjacent area of a substrate.
• the substrate has an attachment area only. In some embodiments the substrate has both an attachment area and an alignment area.
• a substrate with an attachment area only is exemplified in Figure 12.
• one end of each analyte molecule is attached in an area on the substrate where both attachment and alignment will be performed.
• the size of the substrate area, the length of the analyte molecules and the target spacing for the nucleic acid molecules determine the amount of nucleic acid molecules in the liquid being contacted with the substrate. The longer the molecules, the wider the target spacing required to minimize molecule overlap downstream of the alignment forces.
• Step #1 controlled attachment of one end of the molecule and/or linker, is performed as described previously.
• Steps #2 & #3 molecular alignment and attachment of the full molecule, are also performed as previously described.
• the nucleic acids can be attached anywhere on the substrate.
• the number of linkers or contact spot is limiting to the amount of nucleic aid molecules added during the attachment reactions. This is done to limit the amount of unreacted linker, as unreacted linker molecules that are still available may bind the aligned molecules and interfere with the alignment reaction.
• the attachment area comprises both linkers for attachment of the ends of nucleic acids and substrate modifications for the binding of the aligned nucleic acids.
• a substrate with both an attachment and an alignment area is exemplified in Figure 2, with the aligned area indicated by the "direction of alignment” arrow.
• the density of the aligned molecules can be varied by varying the flow direction (See also below).
• the substrate can be functionalized in whole or in part.
• the surface of the substrate can be functionalized to produce a hydrophobic surface using techniques and reagents known in the art (See e.g., Allemand, Bensimon, et al, "pH-Dependent Specific Binding and Combing of DNA, Biophysical Journal, October 1997).
• a hydrophobic surface acts a type of cleaning agent during the method by facilitating the removal (e.g., by washing away) of various salts, proteins etc. apart from the nucleic acid molecules that are attached to the substrate.
• the hydrophobic surface also acts as a cleaning agent by causing hydrophilic contaminants to remain in solution when aligning with one of the meniscus methods known to those of ordinary skill in the art.
• a hydrophobic surface still allows for the binding of DNA either by functionalizing the hydrophobic surface and/or by drying the DNA onto the surface.
• the hydrophobic surface is a amorphous carbon, graphite or similar surface.
• the substrate surface may be functionalized before or after attachment of nucleic acid molecules, e.g., before or after attachment of linkers. Additional aspects of nucleic acid attachment are described in international patent publication WO2007/076132.
• the second step of the methods of the invention is to perform molecular alignment using any of a number of techniques, typically including, but not limited to, fluid flow, and optionally including salinity and temperature control.
• the parts of the nucleic acid molecules (e.g., multi-stranded nucleic acid molecules) other than the end attached to the surface of a substrate will move with a fluid in the direction of fluid flow and therefore be caused to straighten-out downstream of the point of attachment to the substrate.
• the self-organizing properties of nucleic acid molecules will also cause them to align in a substantially parallel manner in a partial monolayer on the surface of the substrate, thereby aiding in the molecular alignment of the molecules.
• the motion characteristics of the fluid movement preferentially are matched to the nucleic acid molecule properties and controllable factors of the first step of the method (attachment to substrate) to create the targeted result.
• the motion characteristics that may be changed include, but are not limited to: the speed, velocity or acceleration of the fluid flow, changes in speed, velocity or acceleration of the fluid flow, directionality of the fluid flow, volume of the fluid and the duration of the fluid flow.
• the fluid used may include, but is not limited to: polar solvents, such as water or alcohol; non-polar solvents, such as acetone; or gases, such as air, nitrogen or argon.
• Fluid types may be may be changed at any time and at any rate during the alignment step.
• the fluid may contain additives in specified amounts including, but not limited to: one or more salts, one or more wetting agents, and/or one or more materials that affect surface tension and/or bonding properties. Fluid additives may be changed at any time and at any rate during the alignment step.
• the fluid temperature is another characteristic that can be controlled and may also be changed at any time and at any rate during the alignment step. Combinations of the fluid motion characteristics, fluid type, fluid additives and fluid temperature may also be used to perform this step.
• a variation of this step of the method is to bind one or more tags to some or all of the molecules to be aligned (including pilot molecules).
• the tag(s) will affect how the molecules behave in the directional fluid flow (or other applied alignment forces, such as electric or magnetic fields) and contribute to the molecular alignment.
• the tag(s) may be bound at the end of molecules which is not attached to the substrate and/or at one or more points along the length of the molecule.
• the size, shape and material composition of the tag(s) are chosen to work with the other elements of the method and all its variations.
• the methods of binding tag(s) are known to those of ordinary skill in the art.
• Another variation of this step of the method uses an electric field as a directional force to generally align molecules, usually in a flow channel (See e.g., PCT published Application WO2007/076132 A2).
• the electrical field may be used other than or in addition to fluid flow and with or without tags.
• the flow channel consists of hydrophobic walls and a hydrophilic membrane lower surface.
• the flow channel may or may not have an upper surface.
• the attached end will stop the molecule movement once the rest of the molecule has generally straightened and aligned in the direction of the electric field.
• the generally aligned molecules are then washed with aqueous solution of declining salinity. The reduced salinity reduces the salt-induced passivation of the phosphate group repulsive forces.
• the increased repulsion of the phosphate groups also increases the repulsive forces between individual molecules.
• This repulsive effect can be modulated with salinity (both species and concentration) to modulate consequent spacing between aligned molecules.
• salinity both species and concentration
• Other variations of this electric field method may be used. The example is meant to be illustrative and not limiting to the techniques envisioned for the invention.
• magnetism as a directional force other than or in addition to fluid flow to perform the molecular alignment. It may be used other than or in addition to fluid flow and with or without tags.
• An example of a technique using magnetism is to attach metallic or magnetic tags or beads to one or more positions on the molecules with methods known to those of ordinary skill in the art including, but not limited to, methods for biological separation or purification techniques using magnetism and attached metallic nano-particles. In this method, engaging the magnetic force in the direction of the intended alignment will pull the metallic tags on the molecules in a fluid toward the magnet but be stopped by the end attached to the substrate. This will create or reinforce a straightening and alignment effect.
• Other variations of this magnetism method may be used. The example is meant to be illustrative and not limiting to the techniques envisioned for the invention.
• alignment is performed by molecular combing, in which there is no flow channel, but the substrate is dipped into solution, allowed to hybridize or otherwise react, and pulled away from solution in a controlled way (See e.g., US patent 6,458,255)
• the materials and techniques used in the second step of the methods may be used to stretch the multi-stranded nucleic acid molecules during alignment and cause them to unwind, partially or completely, from a helix or other natural curved configuration of the nucleic acid molecules and to form a "ladder" configuration of the nucleic acid molecules (Li, M. Q. 1999. Scanning probe microscopy (STM/ AFM) and applications in biology. Applied Physics A. 68, 225-258; See also Figure 13.)
• the techniques for fixing the aligned nucleic acid molecules to the substrate described in third step of the methods may contribute to the creation or maintenance of a ladder configuration.
• a ladder configuration of the nucleic acid molecules will be of particular benefit for any analysis using direct imaging of nucleic acid molecules, especially direct imaging by a TEM of atomic labels on dsDNA.
• the ladder configuration of the nucleic acid molecules facilitates image interpretation and/or improves the quality of the interpretation of the image because the labels are more closely in the same plane as the substrate surface and do not obscure each other.
• the "ladder" configuration is generated by binding the aligned molecules to specific surface.
• the surface is a hydrophobic surface.
• the surface is a graphite surface.
• Aligned molecules can bind to the substrate in a passive mode or through an active mode.
• the aligned molecules can bind the substrate surface upon formation of the alignment.
• the binding to the surface is non-covalent.
• the binding is reversible. This is achieved by one or a combination of the following: slowing and/or stopping the directional fluid flow, changing temperature of the fluid and/or environment, drying the molecules in a directional fashion after fluid flow is stopped, and adding reagents and/or activators that promote binding to the surface before or after the fluid is removed.
• the binding of the aligned molecules to the surface is driven by a variety of factors including solvent composition and surface characteristics.
• the alignment area of the substrate will have a different characteristic than the attachment area resulting in a selective binding of the aligned molecules to the substrate's alignment area.
• the substrate does not have a separate attachment area and the aligned molecules will bind the same area as where the ends of the molecules are attached (e.g., See Fig 12).
• the surface of the substrate may be modified to both allow for the attachment of the end of the molecule (e.g., through linkers) and the binding of the aligned molecules.
• a reaction is performed to attach the aligned molecules to the surface.
• the surface is modified to react with the aligned molecules.
• the surface is modified to selectively react with the backbone of the aligned molecules but not with the end of the aligned molecules. Reactions to attach nucleic acid molecules to the surface are known in the art (See e.g., WO 2007/076132). Any method of attaching the aligned molecules to the surface is embraced by the invention.
• the DNA is elongated when binding the DNA on the surface.
• Methods of elongation nucleic acids are known in the art, for instance a lowering of the surrounding ionic strength will lead to increased intra-molecular repulsion, and elongation of the nucleic acid.
• binding also comprises fixing the aligned molecules to the substrate surface.
• all the molecules are attached to the substrate surface.
• Methods known to the skilled person can be employed in attaching the molecules to the surface of the substrate after alignment, including: UV-crosslinking, ionic attraction between phosphate groups and a functionalized surface (e.g. amino groups, etc.), use of modified DNA to bond to other surface modifications (e.g., nitrogen doping to substrate), cationic surface treatment, or reliance upon van der Waals forces. Additional aspects of nucleic acid attachment are described in international patent publication WO2007/076132.
• the first, second and third steps may be repeated multiple times for the same substrates.
• the sequence and number of steps performed may vary.
• the first step attaching one end of nucleic acid molecules
• the second and third steps are then performed to align these molecules and optionally fix them to a substrate.
• the first step is then performed again, attaching additional DNA molecules to the same substrate or to the first partial monolayer of molecules in the same attachment area(s).
• the second and third steps are then performed again.
• a preferred embodiment of this repetitive step method attaches, aligns and fixes unlabeled DNA molecules (e.g., pilot molecules) in a High Density Spacing pattern on a substrate by carrying out the first, second and third steps.
• a method of fixing is used such that the unlabeled DNA molecules will not become detached when immersed in fluid.
• Labeled DNA molecules are subsequently end-attached and aligned (the first and second steps) in the same attachment area(s).
• the aligned unlabeled DNA molecules (e.g., pilot molecules) on the surface act as guides for improved alignment of the labeled DNA molecules.
• the attachment at one end may occur in an open space on the substrate or on top of the partial monolayer of unlabeled DNA molecules.
• the third step is then performed for the labeled DNA molecules.
• the High Density Spacing of the unlabeled pilot molecules is large enough to allow the labeled DNA molecules to align between the pilot molecules and bind to the surface to form a monolayer with the pilot molecules.
• some or all of the labeled DNA molecules will be attached and/or aligned on top of the unlabeled pilot molecules, forming two or more layers.
• Some variations may use the method to form many layers of aligned molecules.
• the attachment area(s) may be the same or different for each first step and the actual attachment and/or alignment may occur on the substrate surface or on top of other molecule(s).
• Example 1 This example describes the use the method described above for performing molecular alignment in a partial monolayer with High Density Spacing of dsDNA from a sample (sample dsDNA) to be analyzed.
• the sample dsDNA molecules are labeled with are atom(s) having a molecular weight that are detectable in contrast to the background noise on an image from a transmission electron microscope (TEM).
• TEM transmission electron microscope
• the partial monolayer also contains unlabeled dsDNA from a source other than the target sample that separates the labeled dsDNA to improve the quality of the molecular alignment and the subsequent reading of the sample dsDNA molecules.
• the analysis of this preferred embodiment is used to determine the sequence of DNA in contiguous reads, preferably of 5,000 or more, or 10,000 or more, or twenty thousand (20,000) or more base-pairs. Longer and shorter DNA double-strands also are sequenced with this method by using variations of the techniques in the three steps of the method.
• one end of multiple dsDNA molecules are attached to a substrate in a controlled pattern.
• the substrate has one or more thin membrane areas (less than 30 nm thick) where target sample dsDNA will be imaged with a TEM.
• the thin membrane material is designed to cause minimal background noise in the image.
• the pattern of attachment is chosen to result in dsDNA molecules that, post alignment, have a High Density Spacing measurement of under approximately twenty (20) nm.
• This controlled molecule spacing is achieved by mixing universal linkers (linkers that attach to any DNA molecule) at a selected density in a liquid. These universal linkers are then contacted to one or more defined attachment areas on the substrate. The attachment areas cover specific portions of the thin membranes of the substrate.
• the attachment areas Prior to such contact, the attachment areas optionally are prepared to promote binding of the linkers while the surface outside the attachment areas will be prepared to reduce or prevent binding of the linkers.
• the different preparation of selected areas on the substrate may use, but are not limited to, photo lithography techniques combined with photo-sensitive reagents. During contact of the linkers in a liquid to the substrate, diffusion will spread them approximately evenly where they are subsequently allowed or caused to bind to the surface by covalent forces. Any linkers not bound to the surface are then removed. After the linkers are fixed to the substrate, the dsDNA molecules are caused to bind to the linkers in preparation for the alignment step with a method complementary to the universal linkers used.
• the dsDNA molecules are a mixture of labeled target sample dsDNA and unlabeled dsDNA from another source.
• the target sample dsDNA has labels on individual bases in a predetermined schema that can be differentiated in an image taken by a high resolution TEM, such as the labeling described in US patent 7,291,468.
• the unlabeled dsDNA are present in the mixture at a ratio of at least two-to-one (2:1) with the labeled dsDNA.
• the unlabeled dsDNA does not materially obscure the image of the labels on the target sample dsDNA and serve the purpose of separating the labeled dsDNA molecules from each other (acting as spacers) for improved interpretation of the labels. It is expected that interpretation and analysis of the image will allow a determination of the sequence of a high proportion of the target sample dsDNA.
• STEP TWO In the second step, molecular alignment is performed. The parts of the molecules other than the end attached to the surface move with the direction of alignment forces and therefore straighten-out downstream of the point of attachment. Control of salinity, temperature and surface treatments cause the self-organizing properties of multi-stranded nucleic acid molecules to form parallel lines in a partial monolayer on the surface, with controlled spacing. One or more microfluidic channels direct fluid over the substrate.
• Attachment Areas are at the upstream end of one or more thin membranes that are designed to have a length longer than the target sample molecules after alignment.
• the fluid flow optionally is combined with other molecular alignment technique variations including, but not limited to, controlling salinity in a gradient, controlling temperature and using electric field(s) or magnetic field(s) to provide additional force in moving the dsDNA in the direction of the fluid flow.
• STEP THREE
• all of the aligned nucleic acid molecules are fixed to the substrate surface. This is achieved by a combination of slowing the directional fluid flow, drying the dsDNA molecules in a directional fashion as the fluid flow is stopped, and adding reagents and/or activators that create permanent binding to the surface after the fluid is removed.
• Figure 5 illustrates the result of High Density Spacing combined with mixing unlabeled with the labeled molecules to selectively separate the labeled molecules after alignment.
• Figure 5 is the same as Figure 3 but changes the DNA molecule shading to indicate the effect of mixing labeled and unlabeled molecules.
• a substrate with oligonucleotide linkers with sequence A and sequence B is generated according to the routine art.
• Unlabeled 50 kb pilot molecules are prepared according to
• Example 3 below resulting in pilot molecules with a sticky end complementary to sequence A. These pilot molecules are subsequently added to the substrate resulting in the attachment of the pilot molecules to linkers with sequence A. The pilot molecules are next aligned by running a flow stream over the substrate. In the next step the aligned pilot molecules are bound to the substrate by removing the flow stream resulting in a monolayer as depicted in Figure 13.
• Labeled 8-16 kb analyte molecules are prepared according to Example 3 below resulting in analyte molecules with a sticky end with complementary to sequence B. These analyte molecules are subsequently added to the substrate resulting in the attachment of the analyte molecules to linkers with sequence B. Subsequent alignment of the analyte molecules and binding to the substrate results in a pattern wherein the analyte molecules fall within the grid established by the pilot molecules (See Fig 13).
• Genomic DNA was fragmented using a HydroShear® machine (Digilab Genomic Solutions, Ann Arbor, MI) resulting in DNA of about 8,000 to 16,000 basepairs in length.
• the DNA was further processed to remove single strand overhangs using a End-ItTM DNA End-Repair Kit (Epicenter Biotechnologies, Madison, WI). Processing of the DNA resulted in blunt-ended DNA that is 5' phosphorylated.
• the DNA was equipped with adaptamers. Adaptamers were prepared using the following primers:
• Primer ZSlRC has a phosphate on the 5' end.
• the ZSl and ZSlRC primers were mixed together at equal amounts, heated to 95 C and slowly cooled to 4 0 C, resulting in an adaptamer with a non-phosphorylated 5'GGAC overhang on one side, while the adaptamer is blunt ended with a 5' phosphate at the other side.
• the adaptamer was subsequently ligated to the DNA fragments, using a large excess of adaptamers, resulting in a ligation of the blunt end of the adaptamers to the blunt-ended genomic DNA fragments.
• the end result of the ligation is a mixture of DNA fragments with lengths between 8 kb and 16 kb with an additional 45 base pair adaptamer on each end.
• the excess of adaptamers prevents ligation of multiple genomic DNA fragments to each other.
• the DNA fragments were labeled using an amplification reaction.
• ZSlRC was used as a primer and Iodo-U was used in stead of dTTP, resulting in the insertion of labeled U instead of T on the forward strand.
• the amplification mixture was prepared using the following components:
• thermocycler The amplification reaction was run on a thermocycler with the following program:
• YM 100 MICROCON® centrifuge filters (Millipore, Billerica, MA).
• Primer ZlS corresponds to the 18 3 'terminal nucleic acids of primer ZSl .
• the product from the first labeling step was used as a template for the second labeling step.
• ZSlS was used a primer, resulting in the amplification of the reverse strand.
• Bromo-C and Iodo-U were used in place of dCTP & dTTP, respectively.
• This amplification reaction resulted in DNA molecules with one blunt end and one sticky end corresponding to the first 23 nucleotides of ZSlRC.
• the reaction was run only once to generate only double stranded DNA from the excess of single stranded DNA of the first reaction.
• the amplification mixture was prepared using the following components:
• thermocycler This amplification reaction was run on a thermocycler with the following program:
• the resulting product was filtered using YM 100 MICROCON® centrifuge filters (Millipore, Billerica, MA).
• the final product was a double stranded length of labeled DNA with an Iodo label on the forward strand and Bromo and Iodo labels on the reverse strand, and a sticky end on one end of the DNA molecule.
• the reaction was also performed using unlabeled nucleotides resulting in the generation of an unlabeled DNA product with the same length and sticky ends as the labeled DNA.
• the SU-8 mold wafer along with 2-3 drops of the silanizing agent was placed in a Petri dish and put in vacuum desiccator and left under vacuum for at least 1 hour.
• the PDMS pre-polymer was prepared with components in a 10:1 (basexuring agent) ratio
• SYLGARD is a two part resin system containing vinyl groups (part A) and hydrosiloxane groups (part B)).
• the PDMS mixture was subsequently added to the SU-8 master wafer located in the polystyrene Petri dish.
• the PDMS was cured without heating in 24 hours. (Alternatively the PDMS can be cured at 65°C for -1-2 hours; depending on the thickness of the PDMS layer).
• the PDMS mold was peeled from the SU-8 master and cut down to appropriate size.
• the PMDS device has narrow flow channels molded into the bottom surface.
• the PMDS device was placed in contact with a substrate, with the PMDS device providing the walls and "ceiling" of the flow channels, and the substrate providing the "floor,"
• the flow channels are rectangular in cross section and vary from 100 to 300 microns in width and from 50 to 100 microns in height.
• the flow device and substrate are cleaned, and optionally degassed.
• the substrate surface is prepared with a standard, vapor phase silanization protocol, using M-octyl trichlorosilane.
• the PDMS device is then attached to the substrate with minimal pressure.
• 10 uL of the DNA with adaptor in IX TE is introduced at one end of the flow channel and then it is drawn through the flow channel.
• the solution is allowed 5 to 10 minutes for the end-attachment reaction to stabilize.
• the fluid is then moved through the channel with a fluid velocity of approximately 100 microns per second.
• the substrate is used without the flow device, relying on a standard molecular combing protocol whereby the device is submerged in a solution of the DNA and, after 5 to 10 minutes for the end- attachment reaction to stabilize, is withdrawn from the fluid at a velocity of 200-300 microns per second.
• the final product is rinsed with a 70% ethanol solution and several rinsings with purified water.

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