Integrated nucleic acid analysis

• US 8,018,593 B2
• Filed: 04/04/2008
• Issued: 09/13/2011
• Est. Priority Date: 04/04/2007
• Status: Active Grant

• Current Assignee: ANDE Corp.
• Original Assignee: NetBio, Inc.
• Inventors: Thomann, Ulrich Hans, Bogdanov, Valery Leonidovich, Lam, Heung Chuan, Wright, John A., Kellogg, Gregory John, Tan, Eugene, Selden, Richard F.
• Primary Examiner(s): Chowdhury; Tarifur
• Assistant Examiner(s): Lapage; Michael P
• Application Number:
  US12/080,751
• Publication Number:
  US 20090059222A1
• Time in Patent Office: 1,257 Days
• Field of Search: 356/311, 356/317, 356/416, 356/417, 356/344, 422 8207- 8208, 436/172, 2504581-4612, 204600-674
• US Class Current: 356/344
• CPC Class Codes: B01L 2200/0684 Venting, avoiding backpressure, avoid gas bubbles
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/16 Surface properties and coatings
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 : Methods for sequencing
• CPC Class Codes: G01N 2021/6441 : with two or more labels
• CPC Class Codes: G01N 21/6402 : Atomic fluorescence; Laser induced fluorescence
• CPC Class Codes: 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
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6486 : Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2201/06113 : Coherent sources; lasers
• CPC Class Codes: G01N 27/44726 : using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44743 : Introducing samples
• CPC Class Codes: G01N 27/44782 : of a plurality of samples
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The present disclosure provides fully integrated microfluidic systems to perform nucleic acid analysis. These processes include sample collection, nucleic acid extraction and purification, amplification, sequencing, and separation and detection. The present disclosure also provides optical detection systems and methods for separation and detection of biological molecules. In particular, the various aspects of the invention enable the simultaneous separation and detection of a plurality of biological molecules, typically fluorescent dye-labeled nucleic acids, within one or a plurality of microfluidic chambers or channels. The nucleic acids can be labeled with at least 6 dyes, each having a unique peak emission wavelength. The present systems and methods are particularly useful for DNA fragment sizing applications such as human identification by genetic fingerprinting and DNA sequencing applications such as clinical diagnostics.

Claims

1. An integrated biochip system comprising:

(a) a biochip comprising a plurality microfluidic systems, wherein each microfluidic system comprises a first reaction chamber in microfluidic communication with a separation chamber, wherein

the first reaction chamber is adapted for;

(i) nucleic acid extraction;

(ii) nucleic acid purification;

(iii) pre-nucleic acid amplification cleanup;

(iv) nucleic acid amplification;

(v) post-nucleic acid amplification cleanup;

(vi) pre-nucleic acid sequencing cleanup;

(vii) nucleic acid sequencing;

(viii) post-nucleic acid sequencing cleanup;

(ix) reverse transcription;

(x) pre-reverse transcription cleanup;

(xi) post-reverse transcription cleanup;

(xii) nucleic acid ligation;

(xiii) nucleic acid hybridization;

or

(xiv) quantification; and

the separation chamber comprises a detection position; and

(b) a separation and detection system comprising,

(i) a separation element for simultaneously separating a plurality of target analytes in the separation chamber;

(ii) one or more light sources positioned to illuminate the detection positions on the biochip;

(iii) a mirror to scan said one or more light sources sequentially between said detection positions;

(iv) one or a plurality of first optical elements positioned for collecting and directing light emanating from the detection positions; and

(v) a light detector positioned to accept light directed from the one or plurality of first optical elements, wherein the light detector comprises a wavelength dispersive element to disperse the light from the one or plurality of first optical elements according to light wavelength into at least 6 wavelength components and, the wavelength dispersive element is positioned to provide at least a portion of the dispersed at least 6 wavelength components to at least 6 detection elements, wherein

each of the detection elements is in communication with a first control element for simultaneously collecting detection information from each of the detection elements; and

wherein said light detector detects fluorescence from at least 6 dyes labeled to one or more biological molecules, each dye having a unique peak wavelength.

2. The integrated biochip system of claim 1, wherein the first reaction chamber is adapted for nucleic acid extraction.

3. The integrated biochip system of claim 1, wherein the first reaction chamber is adapted for nucleic acid purification.

4. The integrated biochip system of claim 1, wherein the first reaction chamber is adapted for nucleic acid amplification.

5. The integrated biochip system of claim 1, wherein the first reaction chamber is adapted for cleanup.

6. The integrated biochip system of claim 1, wherein the first reaction chamber is adapted for nucleic acid sequencing.

7. The integrated biochip system of claim 1, wherein the first reaction chamber is adapted for reverse transcription.

8. The integrated biochip system of claim 1, wherein the first reaction chamber is adapted for nucleic acid ligation.

9. The integrated biochip system of claim 1, wherein the first reaction chamber is adapted for nucleic acid hybridization.

10. The integrated biochip system of claim 1, wherein the first reaction chamber is adapted for quantification.

11. An integrated biochip system comprising:

(a) a biochip comprising a plurality microfluidic systems, wherein each microfluidic system comprises a first reaction chamber in microfluidic communication with a separation chamber, wherein

the first reaction chamber is adapted for

(i) nucleic acid extraction;

(ii) nucleic acid purification;

(iii) pre-nucleic acid amplification cleanup;

(iv) nucleic acid amplification;

(v) post-nucleic acid amplification cleanup;

(vi) pre-nucleic acid sequencing cleanup;

(vii) nucleic acid sequencing;

(viii) post-nucleic acid sequencing cleanup;

(ix) reverse transcription;

(x) pre-reverse transcription cleanup;

(xi) post-reverse transcription cleanup;

(xii) nucleic acid ligation;

(xiii) nucleic acid hybridization;

or

(xiv) quantification; and

the separation chamber comprises a detection position; and

(b) a separation and detection system comprising,

(i) a separation element for simultaneously separating a plurality of biological molecules comprising DNA sequences, in the separation chamber;

(ii) one or more light sources positioned to illuminate the detection positions on the biochip;

(iii) a mirror to scan said one or more light sources sequentially between said detection positions;

(iv) one or a plurality of first optical elements positioned for collecting and directing light emanating from the detection positions; and

(v) a light detector positioned to accept light directed from the one or plurality of first optical elements, wherein the light detector comprises a wavelength dispersive element to disperse the light from the one or plurality of first optical elements according to light wavelength into at least 6 wavelength components, and the wavelength dispersive element positioned to provide at least a portion of the dispersed at least 6 wavelength components to at least 6 detection elements, wherein each of the detection elements is in communication with a first control element for simultaneously collecting detection information from each of the detection elements; and

wherein said light detector detects fluorescence from at least 8 dyes labeled to one or more DNA sequences, each dye having a unique peak wavelength, said dyes being members of at least two 4-dye containing subsets, such that said dye sets are capable of detecting at least two DNA sequences in a single channel, wherein the number of dyes is a multiple of four, and the number of DNA sequences to be detected is equal to one quarter of the multiple, such that each of the different dyes is present in only one subset.