Methods and systems for direct sequencing of single dna molecules

Abstract

The invention provides improved methods for sequencing nucleic acids, e.g., for medical applications and biomedical research. The disclosed methods can be applied to rapid personalized medicine, genetic diagnosis, pathogen identification, and sequencing species genomes.

1 . A labeled DNA polymerase, wherein said DNA polymerase comprises at least one FRET donor and at least one FRET acceptor,
 wherein said FRET donor and FRET acceptor are positioned on the DNA polymerase so that when the polymerase adds a nucleotide to the nascent strand of DNA, a distinct FRET signal is generated at least depending on which base (A, C, G, T) is incorporated.   
     
     
         2 . The labeled DNA polymerase of  claim 1 , wherein the FRET donor is within 10 angstroms of the Förster radius (R 0 ) from the FRET acceptor when the DNA polymerase is in the open position. 
     
     
         3 . The labeled DNA polymerase of  claim 1 , wherein the Förster radius between the FRET donor and the FRET acceptor changes at least 2.5 angstroms from the open position to the closed position of the DNA polymerase. 
     
     
         4 . The labeled DNA polymerase of  claim 1 , wherein the FRET donor and FRET acceptor are covalently attached to amino acids on a solvent accessible surface of the DNA polymerase. 
     
     
         5 . The labeled DNA polymerase of  claim 1 , wherein the DNA polymerase is from a bacteriophage or a bacteria. 
     
     
         6 . The labeled DNA polymerase of  claim 5 , wherein the bacteriophage is phi-29. 
     
     
         7 . The labeled DNA polymerase of  claim 5 , wherein the bacteria is  E. coli.    
     
     
         8 . The labeled DNA polymerase of  claim 1 , wherein the DNA polymerase is genetically engineered. 
     
     
         9 . The labeled DNA polymerase of  claim 1 , further comprising at least a second FRET donor. 
     
     
         10 . The labeled DNA polymerase of  claim 1 , further comprising at least a second FRET acceptor. 
     
     
         11 . The labeled DNA polymerase of  claim 1 , further comprising at least a second FRET donor and at least a second FRET acceptor. 
     
     
         12 . The labeled DNA polymerase of  claim 1 , wherein the FRET donor and FRET acceptor are positioned so that a distinct FRET signal is generated when the DNA polymerase reads a methylated nucleotide on the template DNA. 
     
     
         13 . The labeled DNA polymerase of  claim 1 , wherein the DNA polymerase is made according to a method comprising the steps of:
 selecting at least one first position on the DNA polymerase to be labeled with a FRET donor and at least one second position on the DNA polymerase to be labeled with a FRET acceptor; and   introducing a non-naturally occurring amino acid at each of the selected positions, thereby making the labeled DNA polymerase.   
     
     
         14 - 24 . (canceled) 
     
     
         25 . A method of sequencing a DNA molecule, said method comprising the steps of:
 a) contacting the labeled DNA polymerase of  claim 1  with a DNA template, wherein said DNA template is hybridized to a primer;   b) adding a DNA sequencing reaction mix under conditions appropriate for DNA polymerization;   c) detecting the identity of each nucleotide incorporated into the new strand of DNA by detecting the FRET signal generated by the labeled DNA polymerase, thereby sequencing the DNA molecule.   
     
     
         26 . The method of  claim 25 , wherein said DNA template is immobilized on a substrate. 
     
     
         27 . The method of  claim 25 , wherein said DNA polymerase is immobilized on a substrate. 
     
     
         28 . The method of  claim 26 , wherein said DNA template is attached to the substrate at more than one site. 
     
     
         29 . The method of  claim 26 , further comprising washing the immobilized DNA template, and repeating steps a)-c). 
     
     
         30 - 38 . (canceled) 
     
     
         39 . A system for real time sequencing of a DNA molecule comprising the labeled DNA polymerase of  claim 1  and optical instrumentation capable of detecting a FRET signal from a single molecule. 
     
     
         40 . (canceled)