Sequencing nucleic acid polymers with electron microscopy
First Claim
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1. A method of achieving consistent base-to-base spacing in a nucleic acid polymer strand comprising:
- a) introducing a portion of a polymer attachment tool into a solution comprising nucleic acid polymer strands;
b) attaching a section of at least one strand to the tool;
c) removing substantially all of the tool with the attached strand from the solution after which at least one region of the strand is suspended between the solution and the tool;
d) depositing the suspended region of the strand onto a substrate, wherein the strand detaches from the tool; and
e) obtaining consistent spacing between consecutive bases of the strand.
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Abstract
This invention relates to using an electron microscope to sequence by direct inspection of labeled, stretched DNA. This method will have higher accuracy, lower cost, and longer read length than current DNA sequencing methods.
95 Citations
37 Claims
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1. A method of achieving consistent base-to-base spacing in a nucleic acid polymer strand comprising:
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a) introducing a portion of a polymer attachment tool into a solution comprising nucleic acid polymer strands; b) attaching a section of at least one strand to the tool; c) removing substantially all of the tool with the attached strand from the solution after which at least one region of the strand is suspended between the solution and the tool; d) depositing the suspended region of the strand onto a substrate, wherein the strand detaches from the tool; and e) obtaining consistent spacing between consecutive bases of the strand. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method of achieving consistent base-to-base spacing in a nucleic acid strand, comprising:
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a) introducing a portion of a polymer attachment tool into a solution comprising nucleic acid polymer strands; b) attaching a section of at least one strand to the tool; c) removing substantially all of the tool with the attached strand from the solution after which at least one region of the strand is suspended between the solution and the tool; d) depositing the suspended region of the strand onto a substrate, wherein the substrate is distinct from the polymer attachment tool; e) obtaining consistent spacing between consecutive bases of the strand.
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22. A method of sequencing a labeled nucleic acid polymer strand, comprising:
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a) introducing a portion of a polymer attachment tool into a solution comprising nucleic acid polymer strands; b) attaching a strand to the tool; c) removing substantially all of the tool with the attached strand from the fluid after which at least one suspended region of the strand is suspended between the fluid and the tool; d) obtaining consistent spacing between consecutive bases in said suspended region of the strand; e) placing the strand on a substrate wherein the substrate is distinct from the polymer attachment tool and such that the consistent spacing is substantially preserved; f) imaging the strand with an electron microscope (EM) to produce EM data; and g) determining the base sequence for substantially the entire strand from the EM data; wherein at least a subset of the bases of the strand are associated with a label that is resolvable by an electron microscope. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)
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Specification