Nucleic acid reading and analysis system

• US 6,403,957 B1
• Filed: 10/16/2000
• Issued: 06/11/2002
• Est. Priority Date: 06/07/1989
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Pirrung, Michael C., Fodor, Stephen P. A., Stryer, Lubert, Read, J. Leighton
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/690,191
• Time in Patent Office: 603 Days
• Field of Search: 250/302, 250/458.1, 435/6, 435/7.1, 536/22.1, 536/25.32
• US Class Current: 506/12
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00434 Liquid crystal masks
• CPC Class Codes: B01J 2219/00436 Maskless processes
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00531 essentially square
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00619 : using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 : by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 : Covalent
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• CPC Class Codes: B01J 2219/00639 : the compounds being trapped in or bound to a porous medium
• CPC Class Codes: B01J 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00695 : Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00711 : Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• 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: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07C 229/14 : to carbon atoms of carbon skeletons containing rings
• CPC Class Codes: C07C 229/16 : to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
• CPC Class Codes: C07D 263/44 : Two oxygen atoms
• CPC Class Codes: C07D 317/62 : with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
• CPC Class Codes: C07H 19/04 : Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/10 : with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/04 : on carriers C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 1/042 : characterised by the nature of the carrier
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 1/062 : for alpha- or omega-carboxy functions
• CPC Class Codes: C07K 17/06 : attached to the carrier via a bridging agent
• CPC Class Codes: C07K 17/14 : Peptides being immobilised on, or in, an inorganic carrier
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C08G 69/00 : Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule products obtained from isocyanates or isothiocyanates C08G18/00; polyamide-imides C08G73/14
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• 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: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: C40B 80/00 : Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• 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: G03F 7/00 : Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor using photoresist structures for special production processes, see the relevant places, e.g. B44C, H01L, e.g. H01L21/00, H05K
• CPC Class Codes: G03F 7/26 : Processing photosensitive materials; Apparatus therefor G03F7/12 - G03F7/24 take precedence
• CPC Class Codes: G03F 7/265 : Selective reaction with inorganic or organometallic reagents after image-wise exposure, e.g. silylation
• CPC Class Codes: G03F 7/38 : Treatment before imagewise removal, e.g. prebaking G03F7/265 takes precedence
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules
• CPC Class Codes: H01L 25/50 : Multistep manufacturing processes of assemblies consisting of devices, each device being of a type provided for in group H01L27/00 or H01L29/00 H01L21/50 takes precedence
• CPC Class Codes: H01L 2924/00 : Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
• CPC Class Codes: H01L 2924/0002 : Not covered by any one of groups H01L24/00, H01L24/00 and H01L2224/00
• CPC Class Codes: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10S 435/961 : including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 435/968 : High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/969 : Multiple layering of reactants
• CPC Class Codes: Y10S 435/973 : Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/807 : Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 428/31971 : Of carbohydrate
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A method and apparatus for preparation of a substrate containing a plurality of sequences. Photoremovable groups are attached to a surface of a substrate. Selected regions of the substrate are exposed to light so as to activate the selected areas. A monomer, also containing a photoremovable group; is provided to the substrate to bind at the selected areas. The process is repeated using a variety of monomers such as amino acids until sequences of a desired length are obtained. Detection methods and apparatus are also disclosed.

Claims

1. A polynucleotide analysis apparatus comprising:

a substrate comprising a plurality of different polynucleotides, each of said different polynucleotides occupying a different location of said substrate, each of said locations having an area of 10<sup>−

1</sup> cm² or less, said substrate comprising more than 10 of such polynucleotides, at least some of said polynucleotides bearing fluorescently labeled target molecules excitable with a laser;

an argon laser light source for directing light at said surface of said substrate, said argon laser lightsource generating light at a frequency to excite said fluorescently labeled target molecules, said frequency different from a frequency of light generated by said fluorescently labeled target molecules;

a lens system to focus light on said substrate;

a beam deflector for directing light to said surface at a right angle to said substrate;

a CCD detector for detecting light fluoresced from said substrate in response to said light from said flurorescently labeled target molecules, said light fluoresced at a wavelength greater than 488 nm;

a translator for scanning light on said substrate whereby a plurality of known locations of said substrate are exposed to light to read a region of said substrate; and

a data collection, recording and management system for storing fluoresced light intensity as a function of location on said substrate, said data collection, recording and management system coupled to said translator and said CCD detector, said data collection, recording and management system storing light intensity as a function of location on said substrate and translating said light intensity as a function of location into nucleic acid sequence information.

2. A polynucleotide analysis apparatus in accordance with claim 1 wherein each location of the substrate is scanned for less than one second.

3. A polynucleotide analysis apparatus comprising:

capillary substrate, said substrate comprising a plurality of different polynucleotides, each of said different polynucleotides occupying a different location of said substrate, each of said locations having an area of 10<sup>−

1</sup> cm² or less, said substrate comprising more than 10 of such polynucleotides, at least some of said polynucleotides bearing fluorescently labeled target molecules excitable with a laser;

an argon laser light source for directing light at a wavelength of about 488 nm or less at of said substrate, said laser generating light at a frequency to excite said fluorescently labeled target molecules, said frequency different from a frequency of light generated by said fluorescently labeled target molecules, said target molecules generating light at a frequency greater than 488 nm;

a lens system to focus light on said substrate;

a beam deflector for directing light to said substrate at a right angle to said substrate, and for separately transmitting fluoresced light from said substrate;

a CCD detector for detecting light fluoresced from said support in response to said light source from said flurorescently labeled target molecules; and

a data collection, recording and management system for storing fluoresced light intensity, said data collection, recording and management system coupled to said CCD detector, said data collection, recording and management system storing light intensity and translating said light intensity into nucleic acid sequence information.

4. A polynucleotide analysis apparatus in accordance with claim 3 wherein each location of the substrate is scanned for less than one second.

5. The apparatus recited in claim 3 wherein said substrate is prepared by spatially addressable light directed synthesis.

6. A polynucleotide analysis apparatus comprising:

a capillary substrate, said substrate comprising a plurality of different polynucleotides, each of said different polynucleotides occupying a different area of said substrate, each of said areas being 10<sup>−

1</sup> cm² or less in detectable area, said substrate comprising more than 10 of such polynucleotides, at least some of said polynucleotides bearing fluorescently labeled target molecules excitable with a laser;

an argon laser light source capable of generating light at a wavelength of about 488 nm or less at said substrate to excite said fluorescently labeled target molecules, which generate light at a frequency greater than 488 nm;

said laser oriented to direct the laser light at said capillaries at an angle capable of contacting said target molecules;

a CCD detector oriented in a manner capable of detecting light fluoresced from said target molecules in said areas; and

a data collection, recording and management system for storing fluoresced light intensity, said data collection, recording and management system coupled to said CCD detector, said data collection, recording and management system storing light intensity and translating said light intensity into nucleic acid sequence information.

7. A polynucleotide analysis apparatus in accordance with claim 6 wherein the substrate is translated relative to the laser light.

8. A polynucleotide analysis apparatus in accordance with claim 6 wherein the substrate is composed of quartz.

9. A polynucleotide analysis apparatus in accordance with claim 6 wherein the substrate has a surface which is a different material than the substrate.

10. A polynucleotide analysis apparatus in accordance with claim 9 wherein the substrate is quartz and the surface is a gel.

11. A polynucleotide analysis apparatus comprising an argon laser light source capable of generating a laser beam providing light at a wavelength of about 488 nm or less to excite fluorescently labeled target molecules attached to said polynucleotides in a substrate to emit excited fluorescent light at a wavelength greater than 488 nm detected by a CCD detector oriented in a manner capable of detecting said emitted fluorescent light from said target molecules irradiated by said laser beam, said substrate having more than 10 polynucleotides and each detectable polynucleotide being present in 10⁻

1 cm² or less in detectable area, the interaction of said laser beam and said polynucleotides being facilitated by a translation system also enabling detection of said fluorescent light by said CCD detector coupled to a data collection, recording and management system for storing fluoresced light intensity and translating said light intensity into nucleic acid sequence information.

12. A polynucleotide analysis apparatus in accordance with claim 11 arranged to examine said polynucleotides referenced with respect to a substrate.

13. A polynucleotide analysis apparatus in accordance with claim 12 wherein said substrate has a surface including a different material than said substrate.

14. A polynucleotide analysis apparatus in accordance with claim 13 wherein said substrate is quartz and said surface includes a gel.

15. A polynucleotide analysis apparatus in accordance with claim 12 wherein said translation system includes a translation table arranged to support said substrate.

16. A polynucleotide analysis apparatus in accordance with claim 15 including a lens system arranged to focus said laser beam onto said substrate.

17. A polynucleotide analysis apparatus in accordance with claim 16 wherein said translation table is an x/y translation table designed to displace said substrate under said lased beam.

18. A polynucleotide analysis apparatus in accordance with claim 16 wherein said substrate is a capillary.