Systems and methods for nucleic acid sequencing

Abstract

The present disclosure provides methods and systems for sequencing nucleic acid molecules. Methods may include sequencing double-stranded nucleic acids or single-stranded nucleic acids. Sequencing may include the use of nucleotides coupled to electrostatic moieties. The electrostatic moieties may be detected by a sensor array. The electrostatic moieties may be reversible electrostatic moieties that are cleaved from the nucleic acid molecule after incorporation of the nucleotide. The electrostatic moieties may be irreversible electrostatic moieties. Nucleotides comprising irreversible electrostatic moieties may be incorporated into the nucleic acid molecule, detected by the sensor array, and exchanged for non-detectable nucleotides.

1 - 58 . (canceled) 
     
     
         59 . A method for detecting a nucleic acid molecule, comprising:
 (a) providing a plurality of single-stranded nucleic acid molecules adjacent to a sensor array, wherein a first single-stranded nucleic acid molecule of said plurality of single-stranded nucleic acid molecules is disposed adjacent to a given sensor of said sensor array;   (b) subjecting said first single-stranded nucleic acid molecule to a nucleic acid incorporation reaction to generate a second single-stranded nucleic acid molecule as a growing strand complementary to said first single-stranded nucleic acid molecule, wherein said nucleic acid incorporation reaction comprises alternately and sequentially (i) incorporating individual nucleotides of a first plurality of nucleotides comprising detectable labels, and (ii) incorporating individual nucleotides of a second plurality of nucleotides that do not comprise detectable labels; and   (c) while or subsequent to conducting said nucleic acid incorporation reaction, using said given sensor to detect signals indicative of a change in charge or conductivity from a double layer comprising said detectable labels, thereby determining a sequence or length of said first single-stranded nucleic acid molecule.   
     
     
         60 . The method of  claim 59 , wherein said first plurality of nucleotides comprises a terminator that prevents an additional nucleotide from stably hybridizing to said first single-stranded nucleic acid molecule. 
     
     
         61 . The method of  claim 59 , wherein said first plurality of nucleotides comprises dideoxynucleotides. 
     
     
         62 . The method of any of  claims 59 - 61 , wherein said second plurality of nucleotides comprises a reversible terminator that prevents an additional nucleotide from stably hybridizing to said first single-stranded nucleic acid. 
     
     
         63 . The method of  claim 59 , wherein said first plurality of nucleotides is exchanged with said second plurality of nucleotides. 
     
     
         64 . The method of any of  claims 59 - 63 , wherein said incorporation of said second plurality of nucleotides corrects phase error by incorporating an individual nucleotide from said second plurality of nucleotides at a location along said first single-stranded nucleic acid molecule in which an individual nucleotide from said first plurality of nucleotides has not been incorporated. 
     
     
         65 . The method of  claim 64 , further comprising continuing said nucleic acid incorporation reaction using said individual nucleotides from said first plurality of nucleotides. 
     
     
         66 . The method of  claim 62 , wherein said reversible terminator is removed subsequent to incorporating said individual nucleotides of said second plurality of nucleotides. 
     
     
         67 . The method of any of  claims 59 - 66 , wherein said detectable labels are electrostatic moieties that are not removable. 
     
     
         68 . The method of any of  claims 59 - 67 , wherein said individual nucleotides of said first plurality of nucleotides include different types of nucleotides, each of which different types of nucleotides is coupled to a single type of detectable label. 
     
     
         69 . The method of any of  claims 59 - 67 , wherein said individual nucleotides of said first plurality of nucleotides include different types of nucleotides, each of which different types of nucleotides is coupled to a different type of detectable label. 
     
     
         70 . The method of  claims 59 - 69 , wherein said given sensor is electrically coupled to a charge double layer comprising said first single-stranded nucleic acid molecule. 
     
     
         71 . The method of  claim 59 , wherein said plurality of single-stranded nucleic acid molecules is coupled to a plurality of beads, wherein said first single-stranded nucleic acid molecule is coupled to a given bead of said plurality of beads, and wherein said charge double layer is adjacent to a surface of said given bead. 
     
     
         72 . The method of  claim 59 , wherein said plurality of single-stranded nucleic acid molecules is coupled to one or more surfaces of said sensor array, wherein said first single-stranded nucleic acid molecule is coupled to a surface of said given sensor, and wherein said charge double layer is adjacent to said surface. 
     
     
         73 . The method of any of  claims 59 - 72 , wherein (b) further comprises providing a priming site adjacent to said first single-stranded nucleic acid and generating said second single-stranded nucleic acid molecule upon primer extension from said priming site. 
     
     
         74 . The method of  claim 73 , wherein said priming site is a self-priming loop. 
     
     
         75 . The method of any of  claims 59 - 74 , wherein said given sensor comprises at least two electrodes. 
     
     
         76 . The method of any of  claims 59 - 75 , wherein said individual nucleotides include different types of nucleotides, and wherein said first single-stranded nucleic acid molecule is brought in contact with said different types of nucleotides sequentially. 
     
     
         77 . The method of  claim 59 , wherein at a given time point during said nucleic acid incorporation reaction, said first single-stranded nucleic acid molecule is brought in contact with individual nucleotides of a first type, and at a subsequent time point during said nucleic acid incorporation reaction, said segment is brought in contact with individual nucleotides of a second type, wherein said first type is different than said second type. 
     
     
         78 . The method of any of  claims 59 - 77 , wherein said individual nucleotides include different types of nucleotides, and wherein said first single-stranded nucleic acid molecule is brought in contact with said different types of nucleotides simultaneously. 
     
     
         79 .- 108 . (canceled)