# US 9,040,237 B2 · Sensor arrays and nucleic acid sequencing applications

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- Number: US 9,040,237 B2
- Document ID: US9040237B2
- Status: Granted
- Kind: B2
- Application number: 11/226,696
- Filed: 2005-09-13
- Published: 2006-09-07
- Granted: 2015-05-26
- Earliest claimed: 2005-03-04
- Assignees: Intel Corporation
- Applicants: Tae-Woong Koo; Selena Chan; Xing Su; Zhang Jingwu; Mineo Yamakawa; Val M. Dubin
- Inventors: Tae-Woong Koo; Selena Chan; Xing Su; Zhang Jingwu; Mineo Yamakawa; Val M. Dubin
- Web page: https://patentgrep.com/patent/US9040237B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- B82Y 30/00: Nanotechnology for materials or surface science, e.g. nanocomposites
- C12Q 1/6869: Methods for sequencing
- G01N 27/4145: {specially adapted for biomolecules, e.g. gate electrode with immobilised receptors}
- G01N 27/4148: {Integrated circuits therefor, e.g. fabricated by CMOS processing}
- B01J 2219/00317: Microwell devices, i.e. having large numbers of wells
- B01J 2219/00511: Walls of reactor vessels
- B01J 2219/00605: the compounds being directly bound or immobilised to solid supports
- B01J 2219/00612: the surface being inorganic
- B01J 2219/00621: by physical means, e.g. trenches, raised areas
- B01J 2219/00626: Covalent
- B01J 2219/0063: Other, e.g. van der Waals forces, hydrogen bonding
- B01J 2219/00641: the porous medium being continuous, e.g. porous oxide substrates
- B01J 2219/00722: Nucleotides
- B01L 3/5085: {for multiple samples, e.g. microtitration plates}
- G01N 27/414: Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

## Abstract

Embodiments of the present invention provide devices methods for sequencing DNA using arrays of reaction cavities containing sensors to monitor changes in solutions contained in the reaction cavities. Additional embodiments provide devices and methods for sequencing DNA using arrays of reaction cavities that allow for optical monitoring of solutions in the reaction cavities. Test and fill reaction schemes are disclosed that allow DNA to be sequenced. By sequencing DNA using parallel reactions contained in large arrays, DNA can be rapidly sequenced.

## Claims

### Claim 1

A method for sequencing DNA, comprising,
providing an array comprised of a plurality of cavities to hold reactants, the cavities having a surface for attachment of DNA and coupled electronic sensors for detecting chemical reaction product changes in a solution, wherein each cavity is coupled to an electronic sensor;
attaching DNA molecules to be sequenced to the surfaces of the cavities so that statistically only one DNA molecule to be sequenced is attached in each cavity;
hybridizing a DNA oligo to each of the DNA molecules to be sequenced, the DNA oligo comprising at its 3′-end a first nuclease-resistant nucleotide or a first nuclease-resistant nucleotide analog;
providing a first set of reactants to the cavities, the reactants comprising:
nucleotides comprising only one type of nucleobases selected from the group consisting of adenine, cytosine, guanine, thymine and uracil;
an enzyme capable of extending at least one DNA molecule, and
an enzyme capable of deconstructing one or more nucleotides from at least one DNA molecule;
wherein said providing reactants to the cavities is done under a condition that at the same time allows the DNA oligo to be extended by a nucleotide that is complementary to a base of the DNA molecules to be sequenced, wherein the base is immediately upstream from the base of the DNA molecules complementary to the first nuclease-resistant nucleotide, and allows the nucleotide to be deconstructed from the DNA oligo;
identifying the base by monitoring increases in products of extension and/or deconstruction with the electronic sensors in the cavities;
attaching the DNA oligo with a second nuclease resistant nucleotide or a second nuclease-resistant nucleotide analog, that is complementary to the base;
repeating said providing reactants to the cavities and determining the identity of the single nucleotide at least one more time; and
determining sequence information for the DNA molecules to be sequenced based on increases in the products of extension and/or deconstruction monitored by the electronic sensors.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

The method of claim 1 wherein the electronic sensors are field effect transistors.

### Claim 5 (depends on 1)

The method of claim 1 wherein the electronic sensors are comprised of carbon nanotubes that are capable of acting as field effect transistors.

### Claim 6 (depends on 4, 5)

The method of claim 4 or 5 wherein monitoring the cavities to detect changes in the reactants comprises detecting a change in current flow in the field effect transistors.

### Claim 7 (depends on 1)

The method of claim 1 wherein the enzyme capable of extending at least one DNA molecule is a polymerase enzyme.

### Claim 8 (depends on 1)

The method of claim 1 wherein the enzyme capable of deconstructing one or more nucleotides from at least one DNA molecule is an exonuclease enzyme.

### Claim 9 (depends on 1)

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

### Claim 10

A method for sequencing DNA, comprising,
providing an array comprised of a plurality of electronic sensors for detecting chemical reaction product changes in a solution, wherein the electronic sensors having a surface for attachment of DNA;
attaching DNA molecules to be sequenced to the surfaces of the electronic sensors so that statistically only one DNA molecule is attached to each of the electronic sensors;
hybridizing a DNA oligo to each of the DNA molecules to be sequenced, the DNA oligo comprising at its 3′-end a first nuclease-resistant nucleotide or a first nuclease-resistant nucleotide analog;
providing a first set of reactants to the electronic sensors, the reactants comprising:
nucleotides comprising only one type of nucleobases selected from the group consisting of adenine, cytosine, guanine, thymine and uracil;
an enzyme capable of extending a DNA molecule, and
an enzyme capable of deconstructing one or more nucleotides from at least one DNA molecule;
wherein said providing reactants to the cavities is done under a condition that at the same time allows DNA oligo to be extended by a nucleotide that is complementary to a base of the DNA molecules to be sequenced, wherein the base is immediately upstream from the base of the DNA molecules complementary to the first nuclease-resistant nucleotide, and allows the nucleotide to be deconstructed from the DNA oligo;
identifying the base by monitoring increases in products of extension and/or deconstruction with the electronic sensors;
attaching the DNA oligo with a second nuclease resistant nucleotide or a second nuclease-resistant nucleotide analog, that is complementary to the base
repeating said providing reactants to the electronic sensors and determining the identity of the one nucleotide at least one time; and
determining sequence information for the DNA molecules to be sequenced based on increases in the products of extension and/or deconstruction monitored by the electronic sensors.

### Claim 11 (depends on 10)

The method of claim 10 wherein the array is comprised of about 2 to about 10,000 electronic sensors.

### Claim 12 (depends on 10)

The method of claim 10 wherein the array is comprised of about 10,000 to about 1,000,000,000 electronic sensors.

### Claim 13 (depends on 10)

The method of claim 10 wherein the electronic sensors are field effect transistors.

### Claim 14 (depends on 10)

The method of claim 10 wherein the electronic sensors are comprised of carbon nanotubes that are capable of acting as field effect transistors.

### Claim 15 (depends on 13, 14)

The method of claim 13 or 14 wherein monitoring the cavities to detect changes in the reactants comprises detecting a change in current flow in the field effect transistors.

### Claim 16 (depends on 10)

The method of claim 10 wherein the enzyme capable of extending at least one DNA molecule is a polymerase enzyme.

### Claim 17 (depends on 10)

The method of claim 10 wherein the enzyme capable of deconstructing one or more nucleotides from at least one DNA molecule is an exonuclease enzyme.

### Claim 18 (depends on 10)

The method of claim 10 wherein attaching DNA molecules to be sequenced to the surfaces of the electronic sensors comprises attaching a DNA probe capable of hybridizing to a sequence present in the DNA molecules to be sequenced to the surface of the electronic sensors and placing the DNA molecules to be sequenced onto the electronic sensors under conditions that allow the DNA molecules to be sequenced to hybridize to the probe DNA.

### Claim 19 (depends on 1)

The method of claim 1, wherein the DNA molecule to be sequenced comprises a poly A tail.

### Claim 20 (depends on 1)

The method of claim 1, wherein determining the sequence information comprises determining a number of repetitive bases in the DNA molecules to be sequenced by an amount of products of extension and/or deconstruction.

### Claim 21 (depends on 10)

The method of claim 10, wherein determining the sequence information comprises determining a number of repetitive bases in the DNA molecules to be sequenced by an amount of products of extension and/or deconstruction.