# US 7,465,578 B2 · Nucleic acid sequencing by Raman monitoring of uptake of precursors during molecular replication

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- Number: US 7,465,578 B2
- Document ID: US7465578B2
- Status: Granted
- Kind: B2
- Application number: 10/886,094
- Filed: 2004-07-07
- Published: 2004-12-09
- Granted: 2008-12-16
- Earliest claimed: 2001-09-24
- Assignees: Intel Corporation
- Inventors: Andrew Berlin; Steven J. Kirch; Gabi Neubauer; Valluri Rao; Mineo Yamakawa
- Web page: https://patentgrep.com/patent/US7465578B2

## CPC

- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- C12Q 1/6869: Methods for sequencing
- G01N 21/65: Raman scattering
- B01L 3/5027: {by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip}
- G01J 3/4406: {Fluorescence spectrometry}
- G01N 21/658: {enhancement Raman, e.g. surface plasmons}
- G01N 2021/6441: {with two or more labels}
- G01N 2021/651: {Cuvettes therefore}
- G01N 2021/653: {Coherent methods [CARS]}
- G01N 2021/655: {Stimulated Raman}
- G01N 2021/656: {Raman microprobe}
- 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)
- 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
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- G01J: MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY (light sources F21, H01J, H01K, H05B; investigating properties of materials by optical means G01N)

## Abstract

The methods, compositions and apparatus disclosed herein are of use for nucleic acid sequence determination. The methods involve isolation of one or more nucleic acid template molecules and polymerization of a nascent complementary strand of nucleic acid, using a DNA or RNA polymerase or similar synthetic reagent. As the nascent strand is extended one nucleotide at a time, the disappearance of nucleotide precursors from solution is monitored by Raman spectroscopy or FRET. The nucleic acid sequence of the nascent strand, and the complementary sequence of the template strand, may be determined by tracking the order of incorporation of nucleotide precursors during the polymerization reaction. Certain embodiments concern apparatus comprising a reaction chamber and detection unit, of use in practicing the claimed methods. The methods, compositions and apparatus are of use in sequencing very long nucleic acid templates in a single sequencing reaction.

## Claims

### Claim 1

An apparatus comprising:
a reaction chamber to contain a polymerase and at least one nucleic acid template attached to an immobilization surface for growing a nascent strand complementary in sequence to the respective template;
a detection unit comprising an excitation source, the detection unit being adapted for identifying and quantifying nucleotide precursors in the reaction chamber to identify nucleotide precursors incorporated into the nascent strand;
a molecule dispenser for dispensing nucleotide precursors into the reaction chamber; and
an information processing and control system operably coupled to the detection unit and the molecule dispenser, for controlling nucleotide precursor release rate from the dispenser to regulate the release of nucleotide precursors by the molecule dispenser to approximately match the rate of incorporation of nucleotide precursors into the nascent strand, wherein the molecule dispenser comprises multiple molecule dispensers for dispensing four types of nucleic acid precursors, whereby the rate of release of the four types of nucleotide precursors is independently controlled.

### Claim 2 (depends on 1)

The apparatus of claim 1, wherein the reaction chamber contains a single nucleic acid molecule attached to an immobilization surface.

### Claim 3 (depends on 1)

The apparatus of claim 1, wherein the reaction chamber contains a silver, gold, platinum, copper or aluminum mesh.

### Claim 4 (depends on 1)

The apparatus of claim 1, further comprising a data storage unit.

### Claim 5 (depends on 1)

The apparatus of claim 1, wherein the reaction chamber and molecule dispenser are part of an integrated chip.

### Claim 6 (depends on 1)

The apparatus of claim 1, wherein the excitation source is a laser.

### Claim 7 (depends on 1)

The apparatus of claim 1, wherein the Raman detector is a spectrometer or a monochromator.