Ph measurement for sequencing of dna

Abstract

The present method involves sequencing by synthesis in which a template strand having an attached primer is immobilized in a small volume reaction mixture. In one embodiment, the reaction mixture is in contact with a sensitive heat sensor, which detects the heat of reaction from incorporation of a complementary base (dNTP) in the presence of appropriate reagents (DNA polymerase, and polymerase reaction buffer). Alternatively, or in addition, a change in pH resulting from the incorporation of nucleotides in the DNA polymerase reaction is measured. A device is provided having delivery channels for appropriate reagents, including dNTPs, which may be delivered sequentially or in a mixture. Preferably, the dNTPs are added in a predetermined sequence, and the dNTP is incorporated or not depending on the template sequence.

1 . A method for obtaining sequence information from a single-stranded DNA template, comprising:
 a) providing a primer region hybridized to the single-stranded DNA template;   b) placing multiple copies of the single-stranded DNA template, including the provided primer region, in a reaction chamber in a microfluidic device comprising a plurality of reaction chambers wherein the reaction chamber holds less than 0.1 μL of reaction mixture;   c) adding to the reaction chamber a mixture containing DNA polymerase and a plurality of nucleotides, allowing incorporation of nucleotides by the DNA polymerase;   d) measuring a pH change in the reaction chamber, said pH change being a result of the incorporation of nucleotides by the DNA polymerase producing a pH change indicative of the incorporation of a complementary nucleotide from the primer region hybridized to the single-stranded DNA template;   e) removing unbound nucleotides from the reaction chamber; and repeating steps c), d), and e) to obtain the sequence information.   
     
     
         2 . The method of  claim 1 , further comprising immobilizing the single-stranded template DNA. 
     
     
         3 . The method of  claim 2 , wherein the immobilizing comprises immobilizing the single-stranded DNA template on a bead. 
     
     
         4 . The method of  claim 3 , wherein the bead is made of metal. 
     
     
         5 . The method of  claim 3 , wherein the bead has magnetic properties. 
     
     
         6 . The method of  claim 1 , wherein the pH change is less than 0.3 pH units. 
     
     
         7 . The method of  claim 1 , wherein the step of measuring pH change is accomplished with a microcantilever sensitive to H+ concentration. 
     
     
         8 . The method of  claim 1 , wherein the step of measuring a pH change comprises measuring a potential difference between a reference electrode and a measuring electrode. 
     
     
         9 . The method of  claim 1 , further comprising adding to a plurality of reaction chambers a single-stranded DNA template that is different in sequence in different reaction chambers. 
     
     
         10 . The method of  claim 9 , wherein the plurality of reaction chambers is contained in a single polymeric substrate provided with fluid channels. 
     
     
         11 . The method of  claim 1 , where the primer region comprises a primer added to the reaction chamber. 
     
     
         12 . The method of  claim 1 , where the reaction chamber is between about 70 picoliters and about 30 femtoliters in volume. 
     
     
         13 . The method of  claim 1 , wherein said multiple copies of the single-stranded DNA template in a reaction chamber comprise at least 10 3  copies. 
     
     
         14 . The method of  claim 1 , wherein the sequence information comprises more than 80 to 200 bases of sequence. 
     
     
         15 . The method of  claim 1 , further comprising the step of splitting pyrophosphate (PPi) to 2Pi. 
     
     
         16 . A microfluidic device for determining DNA sequences, comprising:
 a) a substrate having a plurality of reaction chambers for holding DNA strands, DNA polymerase, and polymerization buffer;   b) fluid channels for delivering dNTPs to the reaction chambers and for removing unincorporated dNTPs from the reaction chambers; and   c) pH sensors operatively associated with individual reaction chambers for detecting pH changes in individual reaction chambers caused by incorporation of the dNTPs.   
     
     
         17 . The device of  claim 16 , further comprising an expandable control line adjacent to an opening in a reaction well for sealing the reaction chambers from the fluid channel. 
     
     
         18 . The microfluidic device of  claim 16 , wherein said pH sensors comprises a microcantilever sensitive to H+ concentration. 
     
     
         19 . The microfluidic device of  claim 16 , wherein said pH sensors comprises a potentiometer for measuring a potential difference between a reference electrode and a measuring electrode.