Nucleic acid sequencing and electronic detection

• US 20100167938A1
• Filed: 12/31/2008
• Published: 07/01/2010
• Est. Priority Date: 12/31/2008
• Status: Active Grant

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Wu, Kai, Su, Xing, Liu, David J.
• Granted Patent: US 8,563,240 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

Embodiments of the present invention provide devices methods for sequencing DNA using arrays of reaction regions containing sensors to monitor changes in solutions or bound molecules contained in the reaction regions. Additional embodiments provide devices and methods for sequencing DNA using arrays of reaction regions that allow for optical monitoring of solutions in the reaction regions. Chemical amplification schemes that allow DNA to be sequenced in which multiple nucleotide addition reactions are performed to detect the incorporation of a base are disclosed. By sequencing DNA using parallel reactions contained in large arrays, DNA can be rapidly sequenced.

Claims

1. ) A method for determining the sequence of a sample of DNA to be sequenced comprising,

providing an array comprised of a plurality of regions, the regions having a surface for attachment of DNA and a coupled sensor for detecting changes in a solution proximal to the region;

attaching DNA to the surfaces of the regions so that statistically one DNA molecule is attached in one region;

hybridizing a primer strand of DNA to the DNA to be sequenced wherein the hybridized primer strand of DNA is terminated with a nuclease-resistant nucleotide;

providing a first set of reactants to the regions comprising a nuclease-resistant nucleotide capable of preventing the extension of a DNA molecule after it is incorporated into the DNA molecule and an enzyme capable of extending a DNA molecule, under conditions that allow the incorporation of the nuclease-resistant nucleotide capable of preventing the extension of a DNA molecule into a hybridized primer strand;

providing a second set of reactants to the regions comprising nucleotides, and an enzyme capable of extending a DNA molecule, under conditions that allow DNA to be extended, wherein the DNA molecule comprising the primer strand is extended by a plurality of nucleotides;

monitoring the regions to detect increases in reaction products in the regions resulting from the extension of the primer strand by a plurality of nucleotides;

removing the plurality of nucleotides that extended the primer strand and the blocking nucleotide;

repeating the elements of terminating the primer strand of DNA, providing a first set of reactants to the regions, providing a second set of reactants to the regions, and monitoring the regions at least one time; and

determining sequence information for the sample of DNA to be sequenced based on increases in reaction products detected in the regions.

2. ) The method of claim 1 wherein the array is comprised of about 2 to about 10,000 regions.

3. ) The method of claim 1 wherein the array is comprised of about 10,000 to about 1,000,000,000 regions.

4. ) The method of claim 1 wherein the sensor is a field effect transistor.

5. ) The method of claim 1 wherein the sensor is comprised of single walled carbon nanotubes that are capable of acting as a field effect transistor.

6. ) The method of claim 3 or 4 wherein monitoring the regions to detect changes in the reactants comprises detecting a change in current flow in a field effect transistor.

7. ) The method of claim 1 wherein the sensor is an electrode.

8. ) The method of claim 1 wherein the enzyme capable of extending a DNA molecule is a polymerase enzyme.

9. ) The method of claim 1 wherein the enzyme capable of deconstructing a DNA molecule is an exonuclease enzyme.

10. ) The method of claim 1 wherein attaching DNA to the surface of regions comprises attaching a DNA probe capable of hybridizing to a sequence present in the sample of DNA to the surface of the region and placing the sample of DNA into the region under conditions that allow DNA in the sample of DNA to hybridize to the probe DNA.

11. ) A method for determining the sequence of a sample of DNA to be sequenced comprising,

providing an array comprised of a plurality of regions, the regions having a surface for attachment of DNA and a coupled sensor for detecting changes in a solution proximal to the region;

attaching DNA to the surfaces of the regions so that statistically one DNA molecule is attached in one region;

hybridizing a primer strand of DNA to the DNA to be sequenced wherein the hybridized primer strand of DNA is terminated with a nuclease-resistant nucleotide;

providing a first set of reactants to the regions comprising blocking nucleotides capable of reversibly preventing the extension of a DNA molecule after incorporation into the DNA molecule and an enzyme capable of extending a DNA molecule, wherein the blocking nucleotides comprise three different bases, under conditions that allow the incorporation of the blocking nucleotides capable of preventing the extension of a DNA molecule into a hybridized primer strand;

providing a second set of reactants to the regions comprising nucleotides, and an enzyme capable of extending a DNA molecule, under conditions that allow DNA to be extended, wherein DNA molecule comprising primer strands are extended by a plurality of nucleotides;

monitoring the regions to detect increases in reaction products in the regions resulting from the extension of the primer strand by a plurality of nucleotides;

removing the plurality of nucleotides that extended the primer strand;

removing the blocking nucleotides;

repeating the elements of terminating the primer strand of DNA, providing a first set of reactants to the regions, providing a second set of reactants to the regions, monitoring the regions, removing the plurality of nucleotides, and removing the blocking nucleotides at least one time; and

determining sequence information for the sample of DNA to be sequenced based on increases in reaction products detected in the regions.

12. ) The method of claim 11 wherein the array is comprised of about 2 to about 10,000 regions.

13. ) The method of claim 11 wherein the array is comprised of about 10,000 to about 1,000,000,000 regions.

14. ) The method of claim 11 wherein the sensor is a field effect transistor.

15. ) The method of claim 11 wherein the sensor is comprised of single walled carbon nanotubes that are capable of acting as a field effect transistor.

16. ) The method of claim 14 or 15 wherein monitoring the regions to detect changes in the reactants comprises detecting a change in current flow in a field effect transistor.

17. ) The method of claim 11 wherein the sensor is an electrode.

18. ) The method of claim 11 wherein the enzyme capable of extending a DNA molecule is a polymerase enzyme.

19. ) The method of claim 11 wherein the enzyme capable of deconstructing a DNA molecule is an exonuclease enzyme.

20. ) The method of claim 11 wherein attaching DNA to the surface of regions comprises attaching a DNA probe capable of hybridizing to a sequence present in the sample of DNA to the surface of the region and placing the sample of DNA into the region under conditions that allow DNA in the sample of DNA to hybridize to the probe DNA.