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) preparing a nicked nucleic acid;   b) providing a sequencing reaction mixture comprising the nicked nucleic acid, a known nucleotide, a nucleic acid polymerase with exonuclease activity in a reaction chamber, wherein the reaction chamber is coupled to a field effect transistor (FET);   c) detecting a signal in the FET in response to excision of a first nucleotide at a first position on the nicked nucleic acid and incorporation of the known nucleotide at a second position on the nicked nucleic acid, thereby determining the identity of a nucleotide complementary to the incorporated known nucleotide in the nicked nucleic acid; and   d) repeating steps b) and c) with additional known nucleotides to determine the identity of a plurality of nucleotides in the nicked nucleic acid.   
     
     
         156 . The method of  claim 155 , wherein preparing the nicked nucleic acid comprises contacting a double-stranded nucleic acid with a uracil DNA glycosylase (UDG), wherein one strand of the double-stranded nucleic acid comprises one or more uracil residues. 
     
     
         157 . The method of  claim 155 , wherein preparing the nicked nucleic acid comprises contacting a double-stranded nucleic acid with a nickase or nicking enzyme, wherein the double-stranded nucleic acid comprises a recognition sequence of the nickase or nicking enzyme. 
     
     
         158 . The method of  claim 155 , wherein preparing the nicked nucleic acid comprises contacting a double-stranded nucleic acid with an enzyme that degrades RNA, wherein one strand of the double-stranded nucleic acid comprises one or more ribonucleotides. 
     
     
         159 . The method of  claim 155 , wherein preparing the nicked nucleic acid comprises providing a double-stranded nucleic acid having a 3′ overhang on one end and hybridizing to the 3′ overhang a nucleic acid that is shorter than the overhang by least one nucleotide. 
     
     
         160 . The method of  claim 155 , wherein preparing the nicked nucleic acid comprises providing a nucleic acid having a double-stranded region and a single-stranded region, wherein the single-stranded region is capable of self-annealing. 
     
     
         161 . The method of  claim 155 , wherein the FET comprises a chemically-sensitive field effect transistor (chemFET). 
     
     
         162 . The method of  claim 161 , wherein the chemFET comprises an ion-sensitive field effect transistor (ISFET) which detects hydrogen ions released from excisions and incorporations associated with the nicked nucleic acid. 
     
     
         163 . A method for sequencing a plurality of nucleic acids, comprising:
 a) providing a plurality of nicked nucleic acids disposed in an array of reaction chambers, wherein the reaction chambers are associated with at least one field effect transistor (FET);   b) contacting the nicked nucleic acids with a known nucleotide and an enzymatic composition having polymerase and exonuclease activity;   c) detecting signals using the at least one FET in response to excisions and incorporations in the plurality of nicked nucleic acids;   d) determining the identity of nucleotides within the nicked nucleic acids on the basis of the detected signals and the identity of the known nucleotide; and   e) repeating steps b) and d) with additional known nucleotides to determine at least a portion of the sequence for the plurality of nicked nucleic acids.   
     
     
         164 . The method of  claim 163 , wherein the reaction chambers have a volume of approximately 1 picoliter (pL) or less. 
     
     
         165 . The method of  claim 163 , wherein an average center-to-center distance between adjacent reaction chambers in the reaction chamber array is less than about 10 μm. 
     
     
         166 . The method of  claim 163 , wherein the array of reaction chambers comprises at least 10 5  reaction chambers. 
     
     
         167 . The method of  claim 163 , wherein the plurality of nicked nucleic acids comprises concatemers of substantially identical nucleic acids. 
     
     
         168 . The method of  claim 163 , wherein the plurality of nicked nucleic acids are coupled to discrete reaction chambers in the array of reaction chambers. 
     
     
         169 . The method of  claim 163 , wherein the at least one FET comprises a chemically-sensitive field effect transistor (chemFET). 
     
     
         170 . The method of  claim 163 , wherein the at least one FET comprises an ion-sensitive field effect transistor (ISFET) configured to detect hydrogen ions released from the excisions and incorporations in the plurality of nicked nucleic acids. 
     
     
         171 . The method of  claim 163 , wherein the enzymatic composition comprises a polymerase with a nucleic acid polymerase with exonuclease activity. 
     
     
         172 . The method of  claim 163 , wherein the enzymatic composition comprises a polymerase and an exonuclease. 
     
     
         173 . A method for sequencing a plurality of nucleic acids, comprising:
 a) providing a plurality of nicked nucleic acids disposed in an array of reaction chambers, wherein the reaction chambers are associated with at least one field effect transistor (FET);   b) contacting the nicked nucleic acids with a known nucleotide and an enzymatic composition having polymerase and exonuclease activity;   c) detecting a signal in the at least one FET in response to excision of a first nucleotide at a first position in the plurality of nicked nucleic acids and incorporation of the known nucleotide at a second position in the plurality of nicked nucleic acids;   d) determining the identity of nucleotides complementary to the incorporated nucleotides within the nicked nucleic acids on the basis of the detected signals and the identity of the known nucleotide; and   e) repeating steps b) and d) with additional known nucleotides to determine at least a portion of the sequence for the plurality of nicked nucleic acids.   
     
     
         174 . The method of  claim 173 , wherein the at least one FET comprises an ion-sensitive field effect transistor (ISFET) which detects hydrogen ions released from excisions and incorporations associated with the nicked nucleic acids. 
     
     
         175 . A composition comprising a nicked nucleic acid, a nucleotide, and one or more polymerases disposed in a reaction chamber capacitively coupled to a field effect transistor (FET). 
     
     
         176 . The composition of  claim 175 , wherein the nicked nucleic acid comprises DNA. 
     
     
         177 . The composition of  claim 175 , wherein the nicked nucleic acid comprises RNA. 
     
     
         178 . The composition of  claim 175 , wherein the nicked nucleic acid is a concatemer of identical nucleic acids. 
     
     
         179 . The composition of  claim 175 , wherein the nicked nucleic acid is further to a microparticle. 
     
     
         180 . The composition of  claim 175 , wherein the composition further comprises a buffering inhibitor. 
     
     
         181 . The composition of  claim 180 , wherein the buffering inhibitor is selected from the group consisting of a phospholipid, a sulfonic acid surfactant, a polyanionic electrolyte or a salt thereof, a polycationic electrolyte or a salt thereof, tetramethyl ammonium and a salt. 
     
     
         182 . The composition of  claim 181 , wherein the buffering inhibitor comprises a combination of two or more different buffering inhibitors. 
     
     
         183 . The composition of  claim 175 , wherein the one or more polymerases comprise  E. coli , DNA polymerase I, Bst DNA polymerase, Taq DNA polymerase, or a combination thereof.