Methods and apparatuses for analyzing polynucleotide sequences

• US 7,462,449 B2
• Filed: 05/14/2004
• Issued: 12/09/2008
• Est. Priority Date: 06/28/1999
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen R.
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US10/845,932
• Publication Number:
  US 20050014175A1
• Time in Patent Office: 1,670 Days
• Field of Search: 435/6, 435/91.1
• US Class Current: 435/6.1
• 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 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/14 Means for pressure control
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: 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 B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: F04B 43/043 Micropumps
• CPC Class Codes: F15C 5/00 Manufacture of fluid circuit elements; Manufacture of assemblages of such elements integrated circuits
• CPC Class Codes: F16K 2099/0074 using photolithography, e.g. etching
• CPC Class Codes: F16K 2099/0076 using electrical discharge machining [EDM], milling or drilling
• CPC Class Codes: F16K 2099/0078 : using moulding or stamping
• CPC Class Codes: F16K 2099/008 : Multi-layer fabrications
• CPC Class Codes: F16K 2099/0084 : Chemistry or biology, e.g. "lab-on-a-chip" technology
• CPC Class Codes: F16K 2099/0094 : Micropumps
• CPC Class Codes: F16K 99/0001 : Microvalves microdevices B81B1/00; manufacture or treatment of devices or systems in or on a substrate B81C1/00; microfluidic structures B01L3/5027; micropumps F04B19/006
• CPC Class Codes: F16K 99/0015 : Diaphragm or membrane valves
• CPC Class Codes: F16K 99/0051 : using electrostatic means
• CPC Class Codes: F16K 99/0059 : actuated by a pilot fluid
• CPC Class Codes: G01N 33/549 : with antigen or antibody entrapped within the carrier

Abstract

Methods for high speed, high throughput analysis of polynucleotide sequences, and apparatuses with which to carry out the methods are provided in the invention.

Claims

1. A method of deriving nucleotide sequence information from polynucleotide molecules, the method comprising the steps of:

immobilizing a plurality of target nucleic acid template molecules on a 2-dimensional substrate, said molecules tagged with a first optically-detectable label attached to each of said molecules;

exposing said molecules to a polymerase and at least one nucleotide comprising a second optically-detectable label under conditions that allow said nucleotide to be incorporated in a primer hybridized to said template molecule if said nucleotide is complementary to a nucleotide in said template immediately 3′

of said primer;

rinsing said substrate to remove unincorporated nucleotides;

positionally locating said second optically-detectable labels appearing in said substrate;

associating each of said positions with a specific polynucleotide in said substrate;

inactivating said second optically-detectable label;

repeating said exposing, rinsing, and locating steps at least once; and

recording a sequence of nucleotides incorporated in said primer at each of said positions in order to determine a sequence of nucleotides in said template,

wherein said substrate is coated with a polyelectrolyte multilayer.

2. The method of claim 1, wherein said exposing step comprises inhibiting incorporation of more than one nucleotide per primer prior to said determining step.

3. The method of claim 1, wherein said immobilizing step comprises depositing said target nucleic acid template molecules onto said substrate.

4. The method of claim 1, wherein said target nucleic acid template molecules are fixed on said substrate by a chemical linkage.

5. The method of claim 1, wherein said substrate comprises glass.