Apparatus and methods for kinetic analysis and determination of nucleic acid sequences

Abstract

A system for determining nucleic acid sequences, including (a) an array of nucleic acid features; (b) a fluidic apparatus configured to deliver sequencing reagents, including polymerases and nucleotides, to the array; (c) a detector configured to obtain pre-equilibrium kinetic measurements from the array at a resolution that distinguishes individual nucleic acid features; (d) a control module having instructions for (i) directing the fluidic apparatus to deliver the sequencing reagents to the array, and (ii) directing the detection apparatus to obtain the kinetic measurements; and (e) an analysis module having instructions for (i) processing the kinetic measurements to determine binding of the polymerase molecules to the nucleic acid features at several pre-equilibrium time points, thereby determining a transient state of the polymerase molecules at the nucleic acid features, and (ii) identifying nucleic acid features that correctly incorporate the nucleotide molecules based on the transient state of the polymerase molecules.

1 - 19 . (canceled) 
     
     
         20 . A system for determining sequences of nucleic acids from pre-equilibrium kinetic measurements of extension reactions for the nucleic acids, the system comprising
 (a) an array comprising nucleic acid features having different nucleotide sequences;   (b) a fluidic apparatus configured to deliver sequencing reagents to the array, wherein the sequencing reagents comprise polymerase molecules and nucleotide molecules for the nucleic acid extensions reactions;   (c) a detection apparatus configured to obtain the kinetic measurements from the array at a resolution that distinguishes individual nucleic acid features of the array;   (d) a control module comprising instructions for
 (i) directing the fluidic apparatus to deliver the sequencing reagents to the array at an initiation time point, and 
 (ii) directing the detection apparatus to obtain the kinetic measurements during the pre-equilibrium time period relative to the initiation time point; and 
   (e) an analysis module comprising instructions for
 (i) processing the kinetic measurements to determine binding of the polymerase molecules to the nucleic acid features at several points during the pre-equilibrium time period, thereby determining a transient state of the polymerase molecules at the nucleic acid features, and 
 (ii) identifying nucleic acid features that correctly incorporate the nucleotide molecules based on the transient state of the polymerase molecules at the nucleic acid features. 
   
     
     
         21 . The system of  claim 20 , wherein the analysis module comprises instructions for
 (i) processing the kinetic measurements to determine pre-steady state kinetic rate profiles of the binding of the polymerase molecules to the nucleic acid features, and   (ii) identifying the nucleic acid features that correctly incorporated the nucleotide molecules, whereby the kinetic rate profiles determine nucleotide molecules that are correctly incorporated into the nucleic acid features.   
     
     
         22 . The system of  claim 21 , wherein the kinetic measurements include data obtained before, during, and after correct incorporation of the nucleotide molecules into the nucleic acid features. 
     
     
         23 . The system of  claim 20 , wherein the detection apparatus is configured to obtain kinetic measurements from the array at a rate of at least 1 signal/second. 
     
     
         24 . The system of  claim 20 , wherein the fluidic apparatus is configured to mix the reagents at a detection site of the array in at least 1 msec. 
     
     
         25 . The system of  claim 20 , wherein the detection apparatus comprises an imaging apparatus. 
     
     
         26 . The system of  claim 25 , wherein the imaging apparatus is configured to obtain images of the array at a rate of at least 1 image/second. 
     
     
         27 . The system of  claim 20 , wherein the array is present in a flow cell and the fluidic apparatus is configured to deliver the sequencing reagents to the flow cell. 
     
     
         28 . The system of  claim 20 , wherein the control module further comprises instructions directing the fluidic apparatus to sequentially deliver sequencing reagents comprising four different nucleotide species, respectively, to the array. 
     
     
         29 . The system of  claim 28 , wherein the control module further comprises instructions to repeat, several times, the sequential delivery of the sequencing reagents comprising four different nucleotide species, respectively, to the array. 
     
     
         30 . The system of  claim 29 , wherein the analysis module comprises instructions to determine the different nucleotide sequences for the nucleic acid features. 
     
     
         31 . The system of  claim 20 , wherein the fluidic apparatus is configured for stopped flow delivery of the sequencing reagents to the array. 
     
     
         32 . The system of  claim 20 , wherein the instructions for processing the signals comprise instructions to determine the transient state from the time duration to reach equilibrium for the binding of the polymerase molecules to the nucleic acid features. 
     
     
         33 . The system of  claim 20 , wherein the instructions for processing the signals comprise instructions to determine the transient state from the binding rate constants for the binding of the polymerase molecules to the nucleic acid features. 
     
     
         34 . The system of  claim 20 , wherein the instructions for processing the signals comprise instructions to determine the transient state from the catalytic rate constants for incorporation of the nucleotide molecules into the nucleic acid features. 
     
     
         35 . The system of  claim 20 , wherein the array comprises a density of at least 100 features/cm 2 . 
     
     
         36 . The system of  claim 20 , wherein the fluidic apparatus is configured to deliver the sequencing reagents to the array prior to the onset of steady state kinetics for the polymerase reaction. 
     
     
         37 . The system of  claim 36 , wherein the fluidic apparatus is configured to deliver fluidic impulses that are confined spatially or temporally.