Electronic sensing for nucleic acid sequencing
Methods for sequencing nucleic acids are presented. Sequencing is accomplished through the detection of a redox active species that is indicative of nucleotide incorporation. In embodiments of the invention, an electrochemical signal indicative of nucleotide incorporation is amplified through cycling before it is detected. Arrays are provided that are capable of massively parallel nucleic acid sequence determination.
1. A method for analyzing a nucleic acid comprising:
providing a nucleic acid molecule to be sequenced,
terminating a complementary nucleic acid polymer hybridized to the nucleic acid molecule to be sequenced with a nuclease resistant nucleotide,
providing a solution comprising a polymerase enzyme, an exonuclease enzyme, and a nucleotide comprising a redox group attached to a phosphate group of the nucleotide, under conditions that allow a complementary nucleotide comprising a redox group to be incorporated into the growing complementary polymer by the polymerase enzyme and excised from the growing complementary strand by the exonuclease enzyme a plurality of times thereby creating a plurality of phosphorylated redox molecules,
removing phosphate groups from the phosphorylated redox molecules that are the product of nucleotide incorporation wherein detachment from the nucleotide and removal of the phosphate group causes the redox molecules to become redox active; and
detecting the presence of de-phosphorylated redox molecules that are a product of the incorporation of a nucleotide triphosphate comprising a redox group into the complementary polymer, wherein detection occurs through the detection of an oxidation or reduction potential for the activated redox molecule.
2. The method of claim 1 wherein the redox group is selected from the group consisting of an aminophenyl, a hydroxyphenyl, and a napthyl.
3. The method of claim 1 wherein the solution comprising a polymerase enzyme, an exonuclease enzyme, and a nucleotide comprising a redox group, also comprises a phosphatase enzyme that is capable of removing phosphate groups from a phosphorylated redox molecule.
4. The method of claim 1 wherein the activated redox molecules are detected through the use of redox cycling.
5. The method of claim 1 wherein the terminating, providing, removing, and detecting are performed a plurality of times and sequence information is determined for a section of the nucleic acid molecule to be sequenced comprising a plurality of bases.
6. A method comprising:
providing an array comprised of a plurality of cavities capable of holding a solution, the plurality of cavities having a surface capable of attaching a DNA molecule and at least two electrodes,
attaching DNA to the surfaces of the cavities so that statistically one DNA molecule is attached in one cavity,
terminating a primer strand of DNA hybridized to the DNA to be sequenced with a nuclease-resistant nucleotide,
providing reactants to the cavities comprising a nucleotide comprising a redox group attached to a terminal phosphate group of the nucleotide, an enzyme capable of extending a DNA molecule, and an enzyme capable of deconstructing a DNA molecule under conditions that allow DNA to be synthesized and deconstructed a plurality of times thereby creating a plurality of phosphorylated redox molecules,
removing phosphate groups from the redox molecules wherein detachment from the nucleotide and removal of the phosphate group causes the redox molecules to become redox active; and
detecting the presence of de-phosphorylated redox molecules that are a product of the incorporation and excision of a nucleotide comprising a redox group into the complementary polymer, wherein detection occurs through the detection of an oxidation or reduction potential for the activated redox molecule.
7. The method of claim 6 wherein the array is comprised of 100 to 10,000 cavities.
8. The method of claim 6 wherein the array is comprised of 10,000 to 1,000,000,000 cavities.
9. The method of claim 6 wherein the enzyme capable of extending a DNA molecule is a polymerase enzyme.
10. The method of claim 6 wherein the enzyme capable of deconstructing a DNA molecule is an exonuclease enzyme.
11. The method of claim 6 wherein the redox group is selected from the group consisting of an aminophenyl, a hydroxyphenyl, and a napthyl.
12. The method of claim 6 wherein the solution comprising a polymerase enzyme, an exonuclease enzyme, and a nucleotide comprising a redox group, also comprises a phosphatase enzyme that is capable of removing phosphate groups from a phosphorylated redox molecule.
13. The method of claim 6 wherein the activated redox molecule is detected through the use of redox cycling.