Sequencing Nucleic Acid Polymers with Electron Microscopy
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.
1 - 93 . (canceled)
94 . A method of providing consistent base-to-base spacing in a polymer, the method comprising:
a) introducing a polymeric binding tool into a fluid composition of the polymer so that the polymer binds to the polymer binding tool; and
b) removing the polymer binding tool from the fluid so that the polymer is stretched into space between an air/fluid interface and the polymer binding tool;
to provide consistent base-to-base spacing in the polymer.
95 . The method according to claim 94 , wherein the polymer is a nucleic acid.
96 . The method according to claim 95 , wherein the base-to-base spacing ranges from 3 to 7 Å.
97 . The method according to claim 95 , wherein the binding tool is dipped into and out of a nucleic acid fluid at a rate ranging from 1 nm/hr to 10 m/s.
98 . The method according to claim 97 , wherein the binding tool is dipped into the fluid at a distance ranging from 1 Å to 20 μm.
99 . The method according to claim 95 , wherein the method comprises depositing the stretched nucleic acid onto a substrate.
100 . The method according to claim 99 , wherein the stretched nucleic acid is deposited onto the substrate using a shelf threading protocol.
101 . The method according to claim 99 , wherein the stretched nucleic acid is deposited onto the substrate using a gap threading protocol.
102 . The method according to claim 99 , wherein the method comprises sequencing the deposited stretched nucleic acid.
103 . The method according to claim 102 , wherein the nucleic acid in the fluid composition is labeled with a contrast agent and the method comprises:
imaging the deposited stretched nucleic acid by electron microscopy to produce electron microscopy (EM) data; and
determining the sequence of the nucleic acid from the EM data.
104 . The method according to claim 103 , wherein the method determines the sequence of 20 or more consecutive bases in the contrast agent labeled nucleic acid.
105 . The method according to claim 104 , wherein the method determines the sequence of 50 or more consecutive bases in the contrast agent labeled nucleic acid.
106 . The method according to claim 103 , wherein the EM data is obtained by transmission electron microscopy.
107 . The method according to claim 103 , wherein the imaging comprises imaging 10,000 or more bases per second.
108 . The method according to claim 103 , wherein the nucleic acid is sequenced at a rate of 1000 bases/second or greater.
109 . A method comprising:
a) introducing a polymer binding tool into a fluid composition of a polymer so that the polymer binds to the polymer binding tool; and
b) removing the polymer binding tool from the fluid so that the polymer is stretched into space between an air/fluid interface and the polymer binding tool.
110 . The method according to claim 109 , wherein the method comprises depositing the stretched polymer onto a substrate.
111 . The method according to claim 109 , wherein the stretched polymer is deposited onto the substrate using a shelf threading protocol.
112 . The method according to claim 109 , wherein the stretched polymer is deposited onto the substrate using a gap threading protocol.
113 . The method according to claim 109 , wherein the polymer is selected from the group consisting of amino acid chains and nucleic acids.