Methods to increase nucleotide signals by raman scattering

Abstract

The methods and apparatus disclosed herein concern nucleic acid sequencing by enhanced Raman spectroscopy. In certain embodiments of the invention, nucleotides are covalently attached to Raman labels before incorporation into a nucleic acid. In other embodiments, unlabeled nucleic acids are used. Exonuclease treatment of the nucleic acid results in the release of labeled or unlabeled nucleotides that are detected by Raman spectroscopy. In alternative embodiments of the invention, nucleotides released from a nucleic acid by exonuclease treatment are covalently cross-linked to nanoparticles and detected by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS). Other embodiments of the invention concern apparatus for nucleic acid sequencing.

1 . A method comprising:
 a) using an exonuclease to release unlabeled nucleotides from one end of one or more nucleic acid molecules;   b) separating the unlabeled nucleotides from the exonuclease and the one or more nucleic acid molecules;   c) identifying the unlabeled nucleotides by Raman spectroscopy to determine identified nucleotides; and   d) determining the sequence of the nucleic acid from the identified nucleotides.   
     
     
         2 . The method of  claim 1 , wherein single molecules of the unlabeled nucleotides are identified by Raman spectroscopy. 
     
     
         3 . The method of  claim 2 , wherein a single nucleic acid molecule is sequenced. 
     
     
         4 . The method of  claim 1 , wherein multiple nucleic acid molecules are sequenced simultaneously. 
     
     
         5 . The method of  claim 1 , wherein the one or more nucleic acid molecules is attached to a surface. 
     
     
         6 . The method of  claim 1 , wherein the unlabeled nucleotides are identified by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS). 
     
     
         7 - 13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the unlabeled nucleotides are attached to nanoparticles after the nucleotides are removed from the nucleic acid. 
     
     
         15 . The method of  claim 1 , wherein the unlabeled nucleotides are attached to nanoparticles before the nucleotides are removed from the nucleic acid. 
     
     
         16 . The method of  claim 15 , wherein nanoparticles are attached to the 3′ end of the nucleic acid. 
     
     
         17 - 21 . (canceled) 
     
     
         22 . A method comprising:
 a) obtaining nucleotides that are attached to Raman labels;   b) synthesizing a nucleic acid comprising labeled nucleotides;   c) removing nucleotides from one end of the nucleic acid;   d) identifying the nucleotides by Raman spectroscopy; and   e) determining the sequence of the nucleic acid.   
     
     
         23 . The method of  claim 22 , wherein single nucleotide molecules are identified by Raman spectroscopy 
     
     
         24 . The method of  claim 22 , wherein each type of nucleotide is labeled with a distinguishable Raman label. 
     
     
         25 . The method of  claim 22 , wherein only pyrimidine nucleotides are labeled with Raman labels. 
     
     
         26 . The method of  claim 22 , further comprising: (i) obtaining at least one template nucleic acid molecule; (ii) hybridizing the template nucleic acid molecule to a primer; and (iii) adding a DNA polymerase to synthesize said nucleic acid. 
     
     
         27 . An apparatus comprising:
 a) a reaction chamber;   b) a microfluidic channel in fluid communication with the reaction chamber;   c) a flow-through cell in fluid communication with the microfluidic channel; and   d) a Raman detection unit operably coupled to the flow-through cell.   
     
     
         28 . The apparatus of  claim 27 , wherein the Raman detector is capable of detecting single molecules of nucleotides. 
     
     
         29 . The apparatus of  claim 28 , wherein the nucleotides are unlabeled. 
     
     
         30 . The apparatus of  claim 27 , further comprising nanoparticles in the flow-through cell.