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 determining the sequence of a nucleic acid, the method comprising:
(a) providing two or more different populations of differentially contrast agent labeled nucleic acid sequences, wherein each of the different populations comprise nucleic acids of identical sequence differentially labeled with the same label;
(b) imaging each of the two or more different populations of differentially labeled nucleic acid sequences by electron microscopy to obtain electron microscopy (EM) data; and
(c) determining the sequence of the contrast agent labeled nucleic acid from the EM data.
95 . The method according to claim 94 , wherein the method determines the sequence of 20 or more consecutive bases in the contrast agent labeled nucleic acid.
96 . The method according to claim 95 , wherein the method determines the sequence of 50 or more consecutive bases in the contrast agent labeled nucleic acid.
97 . The method according to claim 94 , wherein the EM data is obtained by transmission electron microscopy.
98 . The method according to claim 97 , wherein the imaging comprises imaging 10,000 or more bases per second.
99 . The method according to claim 98 , wherein the nucleic acid is sequenced at a rate of 1000 bases/second or greater.
100 . The method according to claim 94 , wherein the method comprises producing the two or more different populations of differentially labeled nucleic acid sequences by producing first and second unlabeled nucleic acid populations contacting each of the first and second populations with a contrast agent that directly labels the nucleic acid to be sequenced to produce the contrast agent labeled nucleic acids.
101 . The method according to claim 100 , wherein the first and second unlabeled populations of nucleic acids are contacted with the same contrast agent under different conditions.
102 . The method according to claim 94 , wherein only a portion of the bases are labeled in the contrast agent labeled nucleic acids.
103 . The method according to claim 94 , wherein the contrast agent labeled nucleic acids are labeled with a high-Z atom contrast agent.
104 . The method according to claim 94 , wherein the deposited contrast agent labeled nucleic acids have a pre-determined configuration.
105 . The method according to claim 104 , wherein the deposited contrast agent labeled nucleic acids have a linear configuration with consistent base-to-base spacing.
106 . The method according to claim 105 , wherein the base-to-base spacing ranges from 3 to 7 Å.
107 . The method according to claim 106 , wherein the providing step comprises:
a) introducing a nucleic acid binding tool into a fluid composition of a contrast agent labeled nucleic acid so that the contrast agent labeled nucleic acid binds to the nucleic acid binding tool;
b) removing the nucleic acid binding tool from the fluid so that the contrast agent labeled nucleic acid is stretched into space between an air/fluid interface and the nucleic acid binding tool; and
c) depositing the stretched contrast agent labeled nucleic acid onto a substrate.
108 . The method according to claim 107 , wherein the stretched contrast agent labeled nucleic acid is deposited onto the substrate using a shelf threading protocol.
109 . The method according to claim 107 , wherein the stretched contrast agent labeled nucleic acid is deposited onto the substrate using a gap threading protocol.