Method and Apparatus for Rapid Nucleic Acid Sequencing

Abstract

Methods and apparatus relating to FET arrays including large FET arrays for monitoring chemical and/or biological reactions such as nucleic acid sequencing-by-synthesis reactions. Some methods provided herein relate to improving signal (and also signal to noise ratio) from released hydrogen ions during nucleic acid sequencing reactions.

1 - 154 . (canceled) 
     
     
         155 . A method for nucleic acid sequencing comprising:
 (a) providing at least one target nucleic acid in a reaction volume, wherein the reaction volume is coupled to a field effect transistor (FET);   (b) providing a reaction mixture to the reaction volume, wherein the reaction mixture comprises a polymerase having an exonuclease activity and a known nucleotide;   (c) incorporating the known nucleotide at a first position on the at least one target nucleic acid and excising a second nucleotide at a second position on the at least one target nucleic acid using the polymerase;   (d) detecting using the FET, one or more hydrogen ions released from incorporation and excision at the first and second positions, respectively;   (e) determining a sequence associated with the first position of the at least one target nucleic acid based on the one or more detected hydrogen ions and the known nucleotide; and   (f) repeating steps b) and e) with additional known nucleotides to determine a sequence for a plurality of additional positions on the at least one target nucleic acid.   
     
     
         156 . The method of  claim 155 , wherein the first position associated with the incorporation of the known nucleotide is adjacent to the second position associated with the excision of the second nucleotide. 
     
     
         157 . The method of  claim 155 , wherein the exonuclease activity of the polymerase comprises a 5′ to 3′ exonuclease activity. 
     
     
         158 . The method of  claim 155 , wherein the polymerase creates a nick in the at least one target nucleic acid excising the second nucleotide at the second position on the target nucleic acid. 
     
     
         159 . The method of  claim 158 , wherein the incorporation of the known nucleotide at the first position on the at least one target nucleic acid incorporates the known nucleotide at a 3′ region of the nick and the excision removes the second nucleotide from a 5′ region of the nick. 
     
     
         160 . The method of  claim 155 , wherein the sequence associated with the first position of the at least one target nucleic acid is discerned without the use of a sequencing primer. 
     
     
         161 . A method for nucleic acid sequencing comprising:
 (a) incorporating a known nucleotide at a first position on a nucleic acid template while excising a second nucleotide at a second position on the nucleic acid template;   (b) detecting one or more hydrogen ions released as a result of the incorporation and excision of step (a);   (c) determining a sequence associated with the first position on the nucleic acid template based on the one or more detected hydrogen ions and the known nucleotide; and   (d) repeating steps (a)-(c) with additional known nucleotides to determine a sequence for a plurality of additional positions on the nucleic acid template.   
     
     
         162 . The method of  claim 161 , wherein the one or more hydrogen ions are detected using an ion-sensitive field effect transistor (ISFET). 
     
     
         163 . The method of  claim 162 , wherein one or more additional nucleic acid templates are substantially simultaneously sequenced using an array of ISFETs. 
     
     
         164 . The method of  claim 161 , wherein step (a) comprises using a polymerase with an exonuclease activity for the incorporation and excision. 
     
     
         165 . The method of  claim 164 , wherein the exonuclease activity of the polymerase comprises a 5′ to 3′ exonuclease activity. 
     
     
         166 . The method of  claim 164 , wherein the polymerase creates a nick in the nucleic acid template. 
     
     
         167 . The method of  claim 166 , wherein the incorporation of the known nucleotide at the first position on the nucleic acid template incorporates the known nucleotide at a 3′ region of the nick and the excision removes the second nucleotide from a 5′ region of the nick. 
     
     
         168 . A method for sequencing a nucleic acid comprising:
 (a) sequentially contacting a nicked nucleic acid with known nucleotides in the presence of a polymerase under conditions suitable for nucleotide incorporation;   (b) detecting byproducts released as a result of incorporation of the known nucleotides in the nicked nucleic acid, wherein the released byproducts indicate nucleotide incorporation of a respective known nucleotide;   (c) discerning a sequence for respective positions of the nucleic acid based on the detected byproducts and the identity of the known nucleotide.   
     
     
         169 . The method of  claim 168 , wherein the byproducts comprise hydrogen ions. 
     
     
         170 . The method of  claim 168 , wherein detecting the byproducts comprises using at least one chemically sensitive field effect transistor (ChemFET). 
     
     
         171 . The method of  claim 168 , wherein detecting the byproducts comprises detecting hydrogen ions using at least one ion-sensitive field effect transistor (ISFET). 
     
     
         172 . The method of  claim 168 , wherein the nicked nucleic acid comprises a concatemer of substantially identical nucleic acids. 
     
     
         173 . The method of  claim 168 , wherein the nicked nucleic acid is coupled to a microparticle. 
     
     
         174 . The method of  claim 168 , wherein detecting the byproducts comprises immobilizing the nicked nucleic acid within a reaction chamber, wherein the reaction chamber is coupled to a sensor.