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 . An apparatus for obtaining sequence information from a single-stranded deoxyribonucleic acid (DNA) template, the apparatus comprising:
 a) a microfluidic device including a plurality of reaction chambers;   b) a microparticle in a particular reaction chamber of the plurality of reaction chambers, the microparticle having multiple copies of the single-stranded DNA template immobilized thereon and with the multiple copies including a primer region hybridized to the single-stranded DNA template;   c) in the particular reaction chamber, a mixture containing DNA polymerase and a plurality of nucleotides and facilitating incorporation of nucleotides by the DNA polymerase;   d) a sensor configured and arranged to detect a signal change in the particular reaction chamber that indicates incorporation of nucleotides by the DNA polymerase, the signal change corresponding to a change in pH indicative of the incorporation of a complementary nucleotide into the primer region hybridized to the single-stranded DNA template and being detected in the particular reaction chamber; and   e) a channel fluidically coupled to the particular reaction chamber and configured and arranged for facilitating removal of unbound nucleotides from the particular reaction chamber, wherein the sequence information is obtained from each of successive detected signal changes, each of which respectively corresponds to incorporation of nucleotides by the DNA polymerase for different mixture reactions in the particular reaction chamber.   
     
     
         2 . The apparatus of  claim 1 , wherein the particular reaction chamber is of sufficiently small volume size for reactants therein to facilitate measurement or sensing of change in heat and/or Hydrogen concentration. 
     
     
         3 . The apparatus of  claim 1 , wherein the particular reaction chamber holds less than 0.1 μL of reaction mixture. 
     
     
         4 . The apparatus of  claim 1 , wherein the sensor is configured to be sensitive to changes in H +  concentration. 
     
     
         5 . The apparatus of  claim 1 , wherein the sensor is configured with a degree of sensitivity that corresponds to a degree of changes in H +  concentration. 
     
     
         6 . The apparatus of  claim 1 , wherein the sensor is configured to detect the signal change such that a degree of the signal change corresponds to a number of the successive changes in H +  concentration.