Quantitative analysis methods on active electronic microarrays

• US 6,492,122 B2
• Filed: 10/10/2001
• Issued: 12/10/2002
• Est. Priority Date: 11/09/2000
• Status: Expired due to Term

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: Wang, Ling, Xu, Xiao, Weidenhammer, Elaine M., Heller, Michael J., Kahl, Brenda F.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/975,408
• Publication Number:
  US 20020150917A1
• Time in Patent Office: 426 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/287.2, 536/22.1, 536/23.7, 536/24-- 2
• US Class Current: 435/6.11
• 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 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00713 Electrochemical synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• 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: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• 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 1/045 using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2565/515 : characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C12Q 2565/607 : being a sensor, e.g. electrode
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• 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: G11C 19/00 : Digital stores in which the information is moved stepwise, e.g. shift registers
• 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

Abstract

The present invention presents techniques useful in methods for gene expression monitoring, and other nucleic acid hybridization assays, that utilize microelectronic arrays to drive the transport and hybridization of nucleic acids. Particularly, methods for normalizing the signals of individual microlocations by the use of an internal control sequence probe are provided. These methods are particularly useful for hybridization assays in which a quantitative comparison of the hybridization of several different sequences at a plurality of microlocations is desired, such as in gene expression analyses.

Claims

1. A method of providing an internal control for an individual test site in a nucleic acid hybridization reaction assay to determine the presence of at least one nucleic acid sequence of interest in at least one nucleic acid containing sample, wherein the nucleic acid hybridization assay is performed on an electronically controlled microarray comprising at least two test sites, the method comprising:

(a) attaching a nucleic acid probe mix consisting of a first nucleic acid probe specific for a first internal control nucleic acid sequence known to be present in the sample, and a second nucleic acid probe specific for a second nucleic acid sequence of interest to a first test site on the electronically controlled microarray;

(b) attaching a mixed nucleic acid probe consisting of the first nucleic acid probe and a third nucleic acid probe specific for a third nucleic acid sequence of interest, wherein the third nucleic acid sequence of interest may be the same as or different than the second nucleic acid sequence of interest, to a second test site on the electronically controlled microarray;

(c) electronically hybridizing the sample nucleic acids from at least one sample to the nucleic acid probes on the first and second test sites;

(d) specifically detecting the extent of hybridization of the sample nucleic acids to the first nucleic acid probe at the first and second test sites;

(e) specifically detecting the extent of hybridization of the sample nucleic acids to the second and third nucleic acid probes at the first and second test sites;

(f) comparing the hybridization values obtained for the first nucleic acid probe at the first and second test sites to obtain a normalization factor; and

(g) normalizing the hybridization values obtained in (e) for the second and third probes using the normalization factor obtained in (f).

2. The method of claim 1 wherein the first nucleic acid sequence is a sequence which encodes, or is complementary to a sequence which encodes, a housekeeping gene.

3. The method of claim 1 wherein the first nucleic acid sequence is an exogenous nucleic acid sequence which has been added to the sample.

4. The method of claim 1 wherein the specific detection in (d) is by detecting a labeled nucleotide which has been specifically incorporated by a nucleic acid polymerase reaction into the sample nucleic acids which contain the first nucleic acid sequence.

5. The method of claim 1, further comprising the step of electronically or passively hybridizing a first reporter nucleic acid comprising a detectable moiety specific for the first nucleic acid sequence to the sample nucleic acids which are hybridized to the first nucleic acid probe at the first and second test sites, wherein the specific detection in (d) is by detecting the detectable moiety.

6. The method of claim 1, further comprising the step of extending the first nucleic acid probe by utilizing the sample nucleic acids which have hybridized to the first nucleic acid probe as a template for a nucleic acid polymerase reaction, wherein the specific detection in (d) is by detecting a labeled nucleotide which has been incorporated into the extended first nucleic acid probe by the polymerase reaction.

7. The method of claim 6, wherein the second and third nucleic acid probes attached in (a) and (b) comprise a protecting group that prevents enzymatic extension of the probes.

8. The method of claim 1 wherein the specific detection in (e) is by detecting a labeled nucleotide which has been specifically incorporated by a nucleic acid polymerase reaction into the sample nucleic acids which contain the second and third nucleic acid sequences of interest.

9. The method of claim 1, further comprising the step of electronically or passively hybridizing one or more reporter nucleic acid probes comprising a detectable moiety to the sample nucleic acids which are hybridized to the second and third nucleic acid probes at the first and second test sites, wherein the reporter probes are specific for the second and third nucleic acid sequences of interest and wherein the specific detection in (e) is by detecting the detectable moiety.

10. The method of claim 1, further comprising the step of extending the second and third nucleic acid probes by utilizing the sample nucleic acids which have hybridized to the second nucleic acid probe as a template for a nucleic acid polymerase reaction, wherein the specific detection in (e) is by detecting a labeled nucleotide which has been incorporated into the extended second and third nucleic acid probes by the polymerase reaction.

11. The method of claim 10, wherein the first nucleic acid probes attached in (a) and (b) comprise a protecting group that prevents enzymatic extension of the probes.

12. The method of claim 1, wherein a first detectable moiety is detected in step (d), and a second detectable moiety is detected in step (e).

13. The method of claim 12 wherein the first and second detectable moieties are independently selected from the group consisting of fluorescent moieties, colorigenic moieties, chemiluminescent moieties, and affinity moieties.

14. The method of claim 13 wherein the first and second detectable moieties are fluorescent moieties.