Sequence determination by direct detection

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.

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         31 . A method for sequencing a molecular sequence comprising: 
 supplying a sequence of nucleotides or nucleotide analogs to a sequencer comprising a polymerase and monomer types for the polymerase, each monomer type having a unique fluorescent tag covalently bonded a portion of the monomer that is released after incorporation of the monomer by the polymerase;    exciting the fluorescent tag with light from an excitation light source;    detecting a property of emitted fluorescent light from a tag before, during and/or after monomer incorporation via a fluorescent light detector; and    converting the detected property into an identity of the incorporated monomer complementary to a nucleotide in the sequence.    
     
     
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         35 . The method of  claim 31 , wherein the detectable property is a duration of the emitted fluorescent light, an intensity of the emitted fluorescent light and/or a frequency spectrum of the emitted fluorescent light.  
     
     
         36 . The method of  claim 35 , wherein the detectable property changes when the monomer is incorporated.  
     
     
         37 . The method of  claim 36 , wherein the change in detectable property occurs after monomer incorporation.  
     
     
         38 . The method of  claim 37 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the β, γ and/or terminal phosphate thereof and where the detectable property is derived from the released portion.  
     
     
         39 . The method of  claim 37 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the β phosphate thereof.  
     
     
         40 . The method of  claim 37 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the γ phosphate thereof.  
     
     
         41 . The method of  claim 37 , wherein the monomers nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the terminal phosphate thereof.  
     
     
         42 . A method for sequencing a molecular sequence comprising: 
 supplying a sequence of nucleotides or nucleotide analogs to a sequencer comprising a polymerase and monomer types for the polymerase, each monomer type having a unique fluorescent tag covalently bonded to a portion of the monomer that is released after incorporation of the monomer by the polymerase;    exciting the fluorescent tag with light from an excitation light source;    detecting a property of emitted fluorescent light before, during and/or after a series of monomer incorporations via a fluorescent light detector; and    converting the detected properties into identities of the series of incorporated monomers complementary to a corresponding nucleotide series in the sequence.    
     
     
         43 . The method of  claim 42 , wherein the detectable property is a duration of the emitted fluorescent light, an intensity of the emitted fluorescent light and/or a frequency spectrum of the emitted fluorescent light.  
     
     
         44 . The method of  claim 43 , wherein the detectable property changes when the monomer is incorporated.  
     
     
         45 . The method of  claim 44 , wherein the change in detectable property occurs after monomer incorporation.  
     
     
         46 . The method of  claim 45 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the β, γ and/or terminal phosphate thereof and where the detectable property is derived from the released portion.  
     
     
         47 . The method of  claim 45 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the β phosphate thereof.  
     
     
         48 . The method of  claim 45 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the γ phosphate thereof.  
     
     
         49 . The method of  claim 45 , wherein the monomers nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the terminal phosphate thereof.