Methods and apparatuses for spatially separating molecules attached to a support and uses in biotechnology

• US 8,021,841 B1
• Filed: 11/24/2008
• Issued: 09/20/2011
• Est. Priority Date: 07/11/2007
• Status: Expired due to Fees

• Current Assignee: Kenneth David Schatz
• Original Assignee: Kenneth David Schatz
• Inventors: Schatz, Kenneth David
• Primary Examiner(s): Whisenant; Ethan C
• Application Number:
  US12/313,763
• Time in Patent Office: 1,030 Days
• Field of Search: 435/6, 536/22.1, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: G01N 30/90 Plate chromatography, e.g. thin layer or paper chromatography

Abstract

Methods and apparatuses for analyzing biomolecules are presented. The methods include dividing of sample molecules into multiple fragments, spatially separating these fragments while maintaining knowledge of their original ordering, and analyzing individual fragments. Apparatuses and methods detailed provide for the spatial separation of sample fragments while maintaining their original ordering. In one embodiment, a sample molecule (2902) is attached to a top surface (2804) of a layer (2802). Layer (2802) is extended such that sample molecule (2902) is divided into fragments (3002), and fragments (3002) are spatially separated. Other embodiments are described and shown.

Claims

1. A method for increasing the spatial separation in a first direction between members of a plurality of elements attached to a first substrate, said elements having an initial ordering along said first direction and said initial ordering maintained comprising,

(a) providing said first substrate, said first substrate including at least one first surfaces, said first surfaces including a plurality of non-intersecting first regions, said first regions having a first ordering along said first direction, and said first substrate including a first means for increasing shortest distances between all pairs of said first regions and maintaining said first ordering along said first direction when acted upon by a first urging,

(b) providing said elements, said elements attached to said first regions with at most one of said elements attached to each member of said first regions, said elements having initial spatial separations along said first direction between adjacent members of said elements of less than approximately 50 microns, said elements containing materials selected from the group consisting of nucleic acid, protein, DNA, double stranded DNA (dsDNA), single stranded DNA (ssDNA), RNA, double stranded RNA (dsRNA), single stranded RNA (ssRNA), DNA/RNA heteroduplexes, mRNA, cDNA, snRNA, siRNA, miRNA, nucleic acid oligomers, labeled-probe-assemblies, nucleic acids with at least one dye-labeled nucleotide, nucleic acid oligomers with at least one dye-labeled nucleotide, nucleic acids with at least one modified nucleotide, nucleic acid oligomers with at least one modified nucleotide, fragments of at least one nucleic acid molecule, islands of amplified nucleic acids, fragments of proteins, denatured proteins, fragments of denatured proteins, protein complexes, and fragments of protein complexes and

(c) applying said first urging to said first means, whereby said elements have increased spatial separations and said initial ordering along said first direction.

2. The method of claim 1 further including,

(d) providing a second substrate, said second substrate including at least one second surfaces, said second surfaces including a plurality of non-intersecting second regions, said second regions having a second ordering along a second direction, and said second substrate including a second means for increasing shortest distances between all pairs of said second regions and maintaining said second ordering along said second direction when acted upon by a second urging,

(e) transferring said elements from said first regions to said second regions and attaching said elements to said second regions, wherein at most one of said elements is attached to each of said second regions and said elements have said initial ordering along said second direction, and

(f) applying said second urging to said second means,

whereby said elements have further increased spatial separations and said initial ordering along said second direction.

3. The method of claim 2 further including, repeating the method of claim 2 for a predetermined number of cycles, said cycles ordered consecutively from cycle number (l) to cycle number (n), and wherein cycle number (m) is an arbitrary member of said cycles, and wherein said second substrate, said second surfaces, said second regions, said second ordering, said second direction, said second means, and said second urging of cycle number (m) are redefined in cycle number (m+1) as said first substrate, said first surfaces, said first regions, said first ordering, said first direction, said first means, and said first urging, respectively, and a new member of said second substrate is provided in step (d) of each member of said cycles.

4. The method of claim 2 wherein transferring said elements from said first regions to said second regions comprises,

(a) providing a film forming solution composed of at least one first solvent and at least one film forming solute, said solute dissolved and/or suspended in said solvent,

(b) covering said elements with a contiguous volume of said solution,

(c) forming a transfer substrate by evaporating said solvent or freezing said solution, wherein said elements are attached to said transfer substrate and have said initial ordering along said first direction,

(d) removing said elements from said first regions, wherein said elements remain attached to said transfer substrate,

(e) contacting and attaching said elements to said second regions, and releasing said elements from said transfer substrate by either, providing a second solvent and dissolving said transfer substrate in said second solvent, or melting said transfer substrate, wherein said elements have said initial ordering in said second direction.

5. The method of claim 2 wherein transferring said elements from said first regions to said second regions comprises,

(a) providing a transfer substrate, said transfer substrate includes a first layer having at least one third surface and said first layer is composed of either, a soluble solid, said soluble solid being soluble in a solvent, a frozen liquid, or a volatile solid compound with sublimation rate measuring greater than 1 micron per 24 hours at temperatures below 300°

C.,

(b) contacting said third surface to said elements such that said elements are attached to said third surface and have said initial ordering along said first direction,

(c) removing said elements from said first regions, wherein said elements remain attached to said third surface,

(d) contacting and attaching said elements to said second regions wherein said elements have said initial ordering along said second direction and,

(e) releasing said elements from said third surface by either, providing said solvent and dissolving said first layer in said solvent, melting said first layer, or sublimating said first layer.

6. The method of claim 5 further including moistening said third surface prior to step (b) with a liquid selected from the group consisting of an aqueous solution, water, buffered water, isopropyl alcohol, ethyl alcohol, said third solvent, polar solvents, non-polar solvents.

7. The method of claim 2 wherein transferring said elements from said first regions to said second regions comprises,

(a) providing a film forming compound composed of materials selected from cyclododecane, camphor, solid compounds with sublimation rates measuring greater than 1 micron per 24 hours at temperatures below 300°

C., and mixtures of these,

(b) forming a transfer substrate, said transfer substrate including at least one layer of said compound and said layer covering and attached to said elements, by either, melting said compound and spreading a melt of said compound over said elements and freezing said melt, or condensing to solid a vapor of said compound over said elements, wherein said elements have said initial ordering along said first direction,

(c) removing said elements from said first regions, wherein said elements remain attached to said transfer substrate,

(d) contacting and attaching said elements to said second regions, and releasing said elements from said transfer substrate by sublimating said layer, wherein said elements have said initial ordering in said second direction.

8. The method of claim 1 further including providing a readout and recording device, processing said elements wherein signals are generated that are correlated with at least one physical property of said elements, and using said readout and recording device to monitor and record said signals.

9. The method of claim 1 wherein multiple independent groups of said elements are processed substantially simultaneously on said substrate.

10. The method of claim 1 further including providing a sample molecule, extending said sample molecule over said first regions approximately along said first direction, attaching said sample molecule to said first regions at multiple locations along said sample molecule, dividing said sample molecule into multiple fragments, wherein said fragments are attached to said first regions with at most one of said fragments attached to each member of said first regions, and said elements comprise said fragments.

11. The method of claim 1 wherein said first substrate is frabricated with stretchable materials selected from the group consisting of elastomeric compounds, plastically deformable compounds, thermoplastic compounds, gels, silicone rubber, fluorosilicone rubber, polyurethane rubber, Sorbothane, EPDM rubber, neoprene rubber, latex, natural rubber, nitrile rubber, butyl rubber, thermoplastic elastomers, sylgard 186, sylgard 184, silicone gel, hydrophobic gels and putties, silicone putty, silicone dilatent compound, blow moldable plastics, low glass transition temperature polymers, mixtures of ethylene-vinyl acetate and ethylene acetate plasticizer and acetone, parafilm M, teflon skived film, polyethylene film, polypropylene film, duco cement, modified nitrocellulose solutions, and mixtures of these.

12. The method of claim 1 wherein at least a portion of said first substrate resembles an approximately planar assembly folded into an accordion folded or pleated structure, and said portion of said substrate has said first surface and a bottom surface and said first surfaces and said bottom surface are approximately parallel and separated by less than one micron and said portion has said accordion folded structure with a plurality of folds wherein each of said folds is aligned approximately parallel to other members of said folds and said folds are aligned approximately perpendicular to said first direction, and wherein said first surface covers convex members of said folds and concave members of said folds and said first regions include the areas of said first surface proximate to said convex members of said folds, and wherein the distances measured along said first direction between said convex members of said folds are increased by applying of said first urging.

17. The method of claim 11 wherein said transfer means comprises,

(a) providing a transfer substrate, said transfer substrate including a first layer having at least one surface which first layer is composed of either, a soluble solid, said soluble solid being soluble in a solvent, a frozen liquid, or a volatile solid compound with sublimation rate measuring greater than 1 micron per 24 hours at temperatures below 300°

C.,

(b) contacting said surface to said elements such that said elements are attached to said surface and have said initial ordering along said first direction,

(c) removing said elements from said first substrate, wherein said elements remain attached to said surface,

(d) contacting and attaching said elements to said second substrate between said second end regions wherein said elements have said initial ordering along said second direction, and

(e) releasing said elements from said surface by providing said solvent and dissolving said first layer in said solvent, melting said first layer, or sublimating said first layer.

13. A method for increasing the spatial separation along a first direction between members of a plurality of elements attached to a first substrate, said elements having an initial ordering along said first direction and said initial ordering maintained comprising,

(a) providing said first substrate, said first substrate including two first end regions, and said first end regions separated from each other along said first direction,

(b) providing said elements, said elements attached to said first substrate between said first end regions, said elements having initial spatial separations along said first direction between adjacent members of said elements less than approximately 50 microns, said elements containing materials selected from the group consisting of nucleic acid, protein, DNA, double stranded DNA (dsDNA), single stranded DNA (ssDNA), RNA, double stranded RNA (dsRNA), single stranded RNA (ssRNA), DNA/RNA heteroduplexes, mRNA, cDNA, snRNA, siRNA, miRNA, nucleic acid oligomers, labeled-probe-assemblies, nucleic acids with at least one dye-labeled nucleotide, nucleic acid oligomers with at least one dye-labeled nucleotide, nucleic acids with at least one modified nucleotide, nucleic acid oligomers with at least one modified nucleotide, fragments of at least one nucleic acid molecule, islands of amplified nucleic acids, fragments of proteins, denatured proteins, fragments of denatured proteins, protein complexes, and fragments of protein complexes and

(c) providing a first means for stretching said first substrate between said first end regions along said first direction,

(d) applying said first means to said first substrate,

whereby said elements have increased spatial separations along said first direction and said initial ordering along said first direction.

14. The method of claim 13 further including,

(e) providing a second substrate, said second substrate including two second end regions, and said second end regions separated from each other along a second direction,

(f) providing a second means for stretching said second substrate between said second end regions along said second direction,

(g) providing a transfer means for transferring said elements from said first substrate to said second substrate between said second end regions and maintaining said initial ordering along said second direction,

(h) applying said transfer means to said elements, and

(i) applying said second means to said second substrate,

whereby said elements have further increased spatial separations and said initial ordering along said second direction.

15. The method of claim 14 further including, repeating the method of claim 14 for a predetermined number of cycles, said cycles ordered consecutively from cycle number (l) to cycle number (n), and wherein cycle number (m) is an arbitrary member of said cycles, and wherein said second substrate, said second end regions, said second direction, and said second means of cycle number (m) are redefined in cycle number (m+1) as said first substrate, said first end regions, said first direction, and said first means, respectively, and a new member of said second substrate is provided in step (e) of each member of said cycles.

16. The method of claim 14 wherein said transfer means comprises,

(a) providing a film forming solution composed of at least one first solvent and at least one film forming solute, said solute dissolved and/or suspended in said first solvent,

(b) covering said elements with a contiguous volume of said solution,

(c) forming a transfer substrate by evaporating said first solvent or freezing said solution, wherein said elements are attached to said transfer substrate and have said initial ordering along said first direction,

(d) removing said elements from said first substrate, wherein said elements remain attached to said transfer substrate,

(e) contacting and attaching said elements to said second substrate between said second end regions and having said initial ordering along said second direction, and releasing said elements from said transfer substrate by either, providing a second solvent and dissolving said transfer substrate in said second solvent, or melting said transfer substrate, wherein said elements have said initial ordering in said second direction.

18. The method of claim 13 wherein said first substrate is fabricated with stretchable materials including elastic materials, elastomeric compounds, plastically deformable compounds, thermoplastic compounds, gels, silicone rubber, fluorosilicone rubber, polyurethane rubber, Sorbothane, EPDM rubber, neoprene rubber, latex, natural rubber, nitrile rubber, butyl rubber, thermoplastic elastomers, Sylgard 186, Sylgard 184, silicone gel, hydrophobic gels and putties, silicone putty, silicone dilatent compound, low-temperature blow moldable plastics, low glass transition temperature polymers, mixtures of ethylene-vinyl acetate and ethylene acetate plasticizer and acetone, Parafilm M, Teflon skived film, polyethylene film, polypropylene film, Duco Cement, modified nitrocellulose solutions, and mixtures of these.

19. The method of claim 13 further including providing a readout and recording device, processing said elements wherein signals are generated that are correlated with at least one physical property of said elements, and using said readout and recording device to monitor and record said signals.

20. The method of claim 13 wherein multiple independent groups of said elements are processed substantially simultaneously on said first substrate.

21. The method of claim 13 further including providing a sample molecule, extending said sample molecule over said first substrate between said first end regions approximately along said first direction, attaching said sample molecule to said first substrate at multiple locations along said sample molecule, dividing said sample molecule into multiple fragments, and said elements comprise said fragments.

22. A method for increasing the spatial separation along a first direction between members of a plurality of elements attached to a first substrate, said elements having an initial ordering along said first direction and said initial ordering maintained comprising,

(a) providing said first substrate, said first substrate including two first end regions, and said first end regions separated from each other along said first direction,

(b) providing said elements, said elements attached to said first substrate between said first end regions,

(c) providing a first means for stretching said first substrate between said first end regions along said first direction,

(d) applying said first means to said first substrate,

(e) providing a second substrate, said second substrate including two second end regions, and said second end regions separated from each other along a second direction,

(f) providing a second means for stretching said second substrate between said second end regions along said second direction,

(g) providing a film forming solution composed of at least one first solvent and at least one film forming solute, said solute dissolved and/or suspended in said first solvent,

(h) covering said elements with a contiguous volume of said solution,

(i) forming a transfer substrate by evaporating said first solvent or freezing said solution, wherein said elements are attached to said transfer substrate and have said initial ordering along said first direction,

(j) removing said elements from said first substrate, wherein said elements remain attached to said transfer substrate,

(k) contacting and attaching said elements to said second substrate between said second end regions and said elements having said initial ordering along said second direction,

(l) releasing said elements from said transfer substrate by either, providing a second solvent and dissolving said transfer substrate in said second solvent, or melting said transfer substrate,

(m) applying said second means to said second substrate, whereby said elements have increased spatial separations and said initial ordering along said second direction, said elements containing materials selected from the group consisting of nucleic acid, protein, DNA, double stranded DNA (dsDNA), single stranded DNA (ssDNA), RNA, double stranded RNA (dsRNA), single stranded RNA (ssRNA), DNA/RNA heteroduplexes, mRNA, cDNA, snRNA, siRNA, miRNA, nucleic acid oligomers, labeled-probe-assemblies, nucleic acids with at least one dye-labeled nucleotide, nucleic acid oligomers with at least one dye-labeled nucleotide, nucleic acids with at least one modified nucleotide, nucleic acid oligomers with at least one modified nucleotide, fragments of at least one nucleic acid molecule, islands of amplified nucleic acids, fragments of proteins, denatured proteins, fragments of denatured proteins, protein complexes, and fragments of protein complexes.

23. The method of claim 22 further including, repeating the method of claim 22 for a predetermined number of cycles, said cycles ordered consecutively from cycle number (l) to cycle number (n), and wherein cycle number (m) is an arbitrary member of said cycles, and wherein said second substrate, said second end regions, said second direction, and said second means of cycle number (m) are redefined in cycle number (m+1) as said first substrate, said first end regions, said first direction, and said first means, respectively, and a new member of said second substrate is provided in step (e) of each member of said cycles.

24. The method of claim 22 wherein said first substrate is frabricated with stretchable materials selected from the group consisting of elastomeric compounds, plastically deformable compounds, thermoplastic compounds, gels, silicone rubber, fluorosilicone rubber, polyurethane rubber, sorbothane, EPDM rubber, neoprene rubber, latex, natural rubber, nitrile rubber, butyl rubber, thermoplastic elastomers, sylgard 186, sylgard 184, silicone gel, hydrophobic gels and putties, silicone putty, silicone dilatent compound, blow moldable plastics, low glass transition temperature polymers, mixtures of ethylene-vinyl acetate and ethylene acetate plasticizer and acetone, parafilm M, teflon skived film, polyethylene film, polypropylene film, duco cement, modified nitrocellulose solutions, and mixtures of these.

25. The method of claim 22 further including providing a readout and recording device, processing said elements wherein signals are generated that are correlated with at least one physical property of said elements, and using said readout and recording device to monitor and record said signals.

26. The method of claim 22 wherein multiple independent groups of said elements are processed substantially simultaneously on said first substrate.

27. The method of claim 22 further including providing a sample molecule, extending said sample molecule over said first substrate between said first end regions approximately along said first direction, attaching said sample molecule to said first substrate at multiple locations along said sample molecule, dividing said sample molecule into multiple fragments, and said elements comprise said fragments.

28. The method of claim 22 wherein said first substrate and said second substrate are fabricated of different materials.