Nucleic acid sequencing by Raman monitoring of molecular deconstruction

Abstract

The disclosed methods, apparatus and compositions are of use for nucleic acid sequencing. More particularly, the methods and apparatus concern sequencing single molecules of single stranded DNA or RNA by exposing the molecule to exonuclease activity, removing free nucleotides one at a time from one end of the nucleic acid, and identifying the released nucleotides by Raman spectroscopy or FRET.

1 - 16 . (canceled)  
     
     
         17 . A method of sequencing nucleic acids comprising: 
 a. preparing a single nucleic acid molecule, one end of the nucleic acid molecule attached to an immobilization surface within a reaction chamber;    b. sequentially removing nucleotides from the unattached end of the nucleic acid molecule;    c. separating the nucleotides from the nucleic acid molecule; and    d. detecting the nucleotides by Raman spectroscopy.    
     
     
         18 . The method of  claim 17 , wherein the nucleotides are removed from the unattached end of the nucleic acid molecule by exonuclease activity.  
     
     
         19 . The method of  claim 17 , wherein a tag molecule is attached to each nucleotide after the nucleotide is removed from the nucleic acid molecule.  
     
     
         20 . The method of  claim 17 , wherein the nucleotides are detected by surface enhanced Raman scattering, surface enhanced resonance Raman scattering, stimulated Raman scattering, inverse Raman, stimulated gain Raman spectroscopy, hyper-Raman scattering or coherent anti-Stokes Raman scattering.  
     
     
         21 . A method of sequencing nucleic acids comprising: 
 a. preparing a single nucleic acid molecule, one end of the nucleic acid molecule attached to an immobilization surface within a reaction chamber;    b. sequentially removing nucleotides from the unattached end of the nucleic acid molecule; and    c. quantifying the nucleotides in the reaction chamber by Raman spectroscopy.    
     
     
         22 . The method of  claim 21 , wherein the nucleotides are removed from the unattached end of the nucleic acid molecule by exonuclease activity.  
     
     
         23 . The method of  claim 21 , wherein a tag molecule is attached to each nucleotide after the nucleotide is removed from the nucleic acid molecule.  
     
     
         24 . The method of  claim 21 , wherein the nucleotides are detected by surface enhanced Raman scattering, surface enhanced resonance Raman scattering, stimulated Raman scattering, inverse Raman, stimulated gain Raman spectroscopy, hyper-Raman scattering or coherent anti-Stokes Raman scattering.  
     
     
         25 . A method of sequencing nucleic acids comprising: 
 a. preparing a single nucleic acid molecule, one end of the nucleic acid molecule attached to an immobilization surface within a reaction chamber;    b. sequentially removing nucleotides from the unattached end of the nucleic acid molecule; and    c. detecting the nucleotides by fluorescence resonance energy transfer (FRET) spectroscopy.    
     
     
         26 . The method of  claim 25 , wherein the nucleotides are attached to acceptor molecules after they are removed from the nucleic acid molecule.  
     
     
         27 . The method of  claim 26 , wherein the nucleotides are removed by an exonuclease.  
     
     
         28 . The method of  claim 27 , wherein one or more donor molecules is attached to the exonuclease.