Method and Apparatus for Rapid Nucleic Acid Sequencing

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.