Sequencing Nucleic Acid Polymers with Electron Microscopy

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.

1 . A method for obtaining sequence information of a DNA polymer strand, said method comprising the steps of:
 a) providing a solution comprising the DNA polymer strand, wherein the DNA polymer strand has been treated such that a plurality of DNA bases have been labeled with a contrast agent with base specificity or base selectivity;   b) introducing a DNA binding tool into the solution and binding a section of the DNA polymer onto the tool;   c) removing the tool from the solution;   d) stretching the labeled DNA polymer strand into space such that the labeled DNA polymer strand is suspended between an air/solvent interface and the tool;   e) depositing the stretched labeled DNA polymer strand onto a substrate;   f) imaging the labeled DNA polymer strand using electron microscopy such that positions of labeled and unlabeled bases are determined; and   g) correlating the positions of labeled and unlabeled bases with the sequence of the DNA polymer.   
     
     
         2 . The method of  claim 1 , wherein the stretched labeled DNA polymer strand is single stranded. 
     
     
         3 . The method of  claim 1 , further comprising the step of denaturing the DNA polymer strand to generate single stranded nucleic acid prior to labeling with the contrast agent. 
     
     
         4 . The method of  claim 3 , wherein the denaturing step is thermal denaturation. 
     
     
         5 . The method of  claim 1 , further comprising the step of depositing a layer of carbon on top of the DNA polymer strand prior to said imaging step. 
     
     
         6 . The method of  claim 1 , wherein the DNA polymer strand is high molecular weight DNA having greater than about 100 kb. 
     
     
         7 . The method of  claim 1 , wherein said stretching step results in consistent base-to-base spacing in the DNA polymer strand. 
     
     
         8 - 9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the length of the suspended region of the DNA polymer strand between the air/solvent interface and the tool is at least about 2 μm. 
     
     
         11 . The method of  claim 1 , wherein only a subset of bases of the DNA polymer strand are labeled. 
     
     
         12 . The method of  claim 11 , wherein the subset of bases comprises thymines and cytosines. 
     
     
         13 . The method of  claim 11 , wherein the subset of bases comprises adenine and guanine. 
     
     
         14 . The method of  claim 1 , wherein the tool in said introducing step is a needle having a tip been functionalized with a first coating that binds to a DNA strand. 
     
     
         15 . The method of  claim 14 , wherein the needle is comprised of a material selected from the group consisting of glass, gold, tungsten, PMMA, polystyrene, PVC, and silicon. 
     
     
         16 . The method of  claim 14 , wherein the first coating is a compound selected from the group consisting of PMMA, polystyrene, PVB, and oligonucleotides. 
     
     
         17 . The method of  claim 14 , wherein the needle is functionalized with a second coating that does not bind to DNA. 
     
     
         18 . The method of  claim 17 , wherein the second coating is a compound selected from the group consisting of octanethiol, hexanethiol, nonanethiol, decanethiol, and septanethiol. 
     
     
         19 . The method of  claim 14 , wherein the tip of the needle has a radius of curvature that is less than about 200 nm. 
     
     
         20 . The method of  claim 14 , wherein the needle is moved into and out of the solution at a rate in a range of about 1 nanometers/second to about 100 meters/second. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the contrast agent comprises a high-Z atom. 
     
     
         23 - 24 . (canceled) 
     
     
         25 . The method of  claim 1 , wherein said depositing step comprises attaching the labeled DNA polymer strand to an imaging substrate by employing shelf threading. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein said depositing step comprises attaching the labeled DNA polymer strand to a support substrate by employing gap threading. 
     
     
         28 . (canceled) 
     
     
         29 . The method of  claim 1 , wherein a plurality of individual DNA polymer strands are stretched into space and deposited onto a substrate and each strand is imaged. 
     
     
         30 . The method of  claim 29 , wherein the plurality of DNA polymer strands are positioned as an array of parallel strands on the substrate. 
     
     
         31 . The method of  claim 1 , further comprising the steps of:
 before step (a), dividing a solution comprising a plurality of DNA polymer strands from the same individual into at least two subsamples, and treating said subsamples so DNA polymer strands are labeled with different base specificity or selectivity; and   after step (g), compiling data obtained from said imaging step of the multiple strands of the DNA polymer to obtain the sequence information.   
     
     
         32 - 93 . (canceled)