Systems and methods for real time single molecule sequence determination

Abstract

A sequencing methodology is disclosed that allows a single DNA or RNA molecule or portion thereof to be sequenced directly and in substantially real time. The methodology involves engineering a polymerase and/or dNTPs with atomic and/or molecular tags that have a detectable property that is monitored by a detection system.

1 . A system for determining sequence information from a single nucleic acid molecule in real time or near real time, comprising:
 at least one target nucleic acid template;   a single-molecule sequencer comprising a polymerase and one or more nucleotides or nucleotide analogs for the polymerase, wherein the polymerase and/or at least one nucleotide or nucleotide analog comprises a tag having a detectable property, and the detectable property of at least one tag changes during polymerization of one or more nucleotides or nucleotide analogs by the polymerase;   an excitation source adapted to excite at least one tag having a detectable property;   a detector adapted to detect one or more changes in the detectable property of at least one tag; and   an analyzer adapted to convert one or more changes in detectable property detected by the detector into an identity of one or more nucleotide bases of the at least one target nucleic acid molecule.   
     
     
         2 . The system of  claim 1 , wherein one or more changes in detectable property of at least one tag results from FRET between the polymerase tag and the tag of at least one nucleotide or nucleotide analog. 
     
     
         3 . The system of  claim 1 , wherein the polymerase tag comprises a Forster resonance energy transfer (FRET) donor. 
     
     
         4 . The system of  claim 1 , wherein at least one nucleotide or nucleotide analog tag comprises a FRET acceptor. 
     
     
         5 . The system of  claim 1 , wherein at least one tag of a nucleotide or nucleotide analog is bonded to a beta (β) phosphate, a gamma (γ) phosphate, or any other terminal phosphate of the nucleotide or nucleotide analog. 
     
     
         6 . The system of  claim 1 , wherein the analyzer converts one or more changes in detectable property detected by the detector into the identity of one or more nucleotide bases of two or more target nucleic acid molecules in parallel. 
     
     
         7 . The system of  claim 1 , wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, SEQUENASE®, and the Klenow fragment from  E. coli  DNA polymerase I, Superscript™ reverse transcriptase, HIV reverse transcriptase, Phi-29 DNA polymerase and mixtures or combinations thereof. 
     
     
         8 . The system of  claim 1 , wherein the polymerase naturally lacks or has been genetically modified to lack 3′ to 5′ exonuclease activity. 
     
     
         9 . The system of  claim 1 , wherein the single molecule sequencer further comprises a surface comprising regions, areas, wells, grooves, channels or patterned arrays wherein the polymerase is immobilized. 
     
     
         10 . The system of  claim 1 , wherein two or more types of nucleotide or nucleotide analog each comprises a tag that is different and detectably discernible from the tag of any other type of nucleotide or nucleotide analog and wherein the tag of each type of nucleotide or nucleotide analog correlates with its base identity. 
     
     
         11 . A system for determining sequence information from a single nucleic acid molecule molecule in real time or near real time, comprising:
 at least one target nucleic acid molecule;   a single-molecule sequencer comprising a polymerase having at least one tag associated therewith;   an excitation source adapted to excite at least one tag on the polymerase;   a detector adapted to detect a response from the at least one excited tag on the polymerase, where the response from the at least one tag changes during polymerization of one or more nucleotides or nucleotide analogs by the polymerase; and   an analyzer adapted to convert one or more responses detected by the detector into an identity of one or more nucleotide bases of the at least one target nucleic acid molecule.   
     
     
         12 . The system of  claim 11 , wherein the polymerase tag comprises a Forster resonance energy transfer (FRET) donor. 
     
     
         13 . The system of  claim 11 , wherein one or more responses detected by the detector is a result of FRET between the polymerase tag and a tag of at least one nucleotide or nucleotide analog polymerized by the polymerase. 
     
     
         14 . The system of  claim 11 , wherein the analyzer converts one or more changes in detectable property detected by the detector into the identity of one or more nucleotide bases of two or more target nucleic acid molecules in parallel. 
     
     
         15 . The system of  claim 11 , wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, SEQUENASE®, and the Klenow fragment from  E. coli  DNA polymerase I, Superscript™ reverse transcriptase, HIV reverse transcriptase, Phi-29 DNA polymerase and mixtures or combinations thereof. 
     
     
         16 . The system of  claim 11 , wherein the polymerase naturally lacks or has been genetically modified to lack 3′ to 5′ exonuclease activity. 
     
     
         17 . The system of  claim 11 , wherein the single molecule sequencer further comprises a surface comprising regions, areas, wells, grooves, channels or patterned arrays wherein the polymerase is immobilized.