PH MEASUREMENT FOR SEQUENCING OF DNA
First Claim
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 μ
L 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.
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Accused Products
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.
90 Citations
19 Claims
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1. A method for obtaining sequence information from a single-stranded DNA template, comprising:
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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 μ
L 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A microfluidic device for determining DNA sequences, comprising:
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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. - View Dependent Claims (17, 18, 19)
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Specification