PH MEASUREMENT FOR SEQUENCING OF DNA

摘要

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.

主权项

1. A method for sequencing a nucleic acid sample, comprising:
(a) providing a plurality of nucleic acid molecules and a polymerase in one or more thermally isolated chambers among a plurality of thermally isolated chambers that each has a volume less than about 0.1 μL, wherein each of said plurality of nucleic acid molecules comprises (i) a nucleic acid sequence of a single-stranded nucleic acid template derived from said nucleic acid sample, and (ii) a primer hybridized thereto, which primer comprises a hairpin formed by self-hybridization or a single-stranded oligonucleotide that has sequence complementarity with said single-stranded nucleic acid template; (b) providing a plurality of nucleotides of a single species in said one or more thermally isolated chambers comprising said plurality of nucleic acid molecules; (c) detecting signal(s) indicative of an enthalpy change in said one or more thermally isolated chambers comprising said plurality of nucleic acid molecules, wherein said enthalpy change is associated with incorporation of one or more nucleotides of said plurality of nucleotides of said single species by said polymerase into a given one of said plurality of nucleic acid molecules to yield a nucleic acid strand that has sequence complementarity with said single-stranded nucleic acid template; and (d) repeating (b) and (c) to obtain a sequence of said single-stranded nucleic acid template.