US2008014592A1PendingUtilityA1

Nucleic Acid Sequence Analysis

67
Individually held — no corporate assignee on recordPriority: Jul 28, 1997Filed: Aug 3, 2007Published: Jan 17, 2008
Est. expiryJul 28, 2017(expired)· nominal 20-yr term from priority
Inventors:Daniel Densham
C12Q 1/6825C12Q 1/6869C12Q 1/68
67
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Claims

Abstract

The present invention relates to a method for determining the sequence of a polynucleotide, the method comprising the steps of: (i) reacting a target polynucleotide with a polymerase enzyme immobilized on a solid support, and the different nucleotides, under conditions sufficient for the polymerase reaction; and (ii) detecting the incorporation of a specific nucleotide complementary to the target polynucleotide, by measuring radiation.

Claims

exact text as granted — not AI-modified
1 . A method for sequencing a polynucleotide, comprising the steps of: 
 (i) reacting a target polynucleotide with a polymerase, wherein said polymerase is immobilized on a solid support, and nucleotides, under conditions sufficient for the polymerase reaction; and    (ii) detecting the interaction between the polymerase, the target polynucleotide, and each specific nucleotide complementary to the target polynucleotide that is incorporated into a nascent polynucleotide being synthesized as a result of the polymerase reaction, to thereby determine the sequence of the target polynucleotide, the detection being carried out by measuring a change in or absorption of radiation that occurs during the interaction.    
     
     
         2 . The method according to  claim 1 , wherein steps (i) and (ii) are conducted with each of the complementary nucleotides in turn, until incorporation is detected, and then repeated.  
     
     
         3 . The method according to  claim 1 , wherein step (i) is conducted with all the complementary nucleotides present.  
     
     
         4 . The method according to  claim 1 , wherein the nucleotides comprise a 3′ blocking group which is removed after the polymerase reaction.  
     
     
         5 . The method according to  claim 4 , wherein the blocking group is selectively removed by pulsed monochromatic light.  
     
     
         6 . The method according to  claim 4 , wherein the nucleotides comprise a further blocking group at the terminal phosphate group of the triphosphate chain, and the further blocking group is removed prior to the removal of the 3′ blocking group.  
     
     
         7 . The method according to  claim 6 , wherein the further blocking groupies selectively removed by pulsed monochromatic light under conditions different from those required to remove the 3′ blocking group.  
     
     
         8 . The method according to  claim 7 , wherein the further blocking group is removed by pulsing the monochromatic light for a duration different from that required to remove the 3’ blocking group.  
     
     
         9 . The method according to  claim 1 , wherein step (i) further comprises introducing a competitive inhibitor of the polymerase.  
     
     
         10 . The method according to  claim 1 , wherein the target polynucleotide of step (i) is bound to the polymerase enzyme by a β 2  dimer complex.  
     
     
         11 . The method according to  claim 1 , wherein the polymerase is  E. coli  DNA polymerase III or T7 polymerase.  
     
     
         12 . The method according to  claim 1 , wherein the polymerase is a Taq polymerase.  
     
     
         13 . The method according to  claim 1 , wherein the polymerase is reverse transcriptase.  
     
     
         14 . The method according to  claim 1 , wherein step (ii) comprises detection of a change in resonance signal over time.  
     
     
         15 . The method according to  claim 1 , wherein the radiation is electromagnetic.  
     
     
         16 . The method according to  claim 15 , wherein the electromagnetic radiation is in the infra-red spectrum.  
     
     
         17 . The method according to  claim 1 , wherein the incorporation of a nucleotide is detected using NMR.  
     
     
         18 . The method according to  claim 1 , wherein the polynucleotide is DNA.  
     
     
         19 . The method according to  claim 1 , wherein measurement is carried out using evanescent wave spectroscopy.  
     
     
         20 . The method according to  claim 1 , wherein the nucleotides are not labeled.  
     
     
         21 . The method according to  claim 1 , wherein the effect detected results from a conformation or mass change of the polymerase that occurs upon incorporation of the nucleotide.

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