Process for specimen handling for analysis of nucleic acids

• US 5,436,129 A
• Filed: 10/12/1993
• Issued: 07/25/1995
• Est. Priority Date: 11/17/1989
• Status: Expired due to Fees

• Current Assignee: Gene Tec Corporation
• Original Assignee: Gene Tec Corporation
• Inventors: Stapleton, Marilyn J.
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/135,131
• Time in Patent Office: 651 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/287, 435/299, 436/807, 436/820, 436/177, 436/178, 436/515, 436/516, 422/56, 422/58, 422/61, 422/69, 422/101, 422/102, 422/104, 204/182.6, 204/182.8, 204/182.9, 204/299 R, 935/76, 935/77
• US Class Current: 435/6.14
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2300/043 Hinged closures
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/0822 Slides
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 3/5023 with a sample being transported to, and subsequently stored in an absorbent for analysis
• 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 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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: B01L 9/527 for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: G01N 1/31 Apparatus therefor
• CPC Class Codes: G01N 1/312 for samples mounted on planar substrates
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

A process for handling a biological specimen for the analysis of nucleic acid sequences wherein the biological specimen is immobilized within a carrier device for controlled temperature conditions and the sequential addition of fluid treatments. The fluid treatments arm selected from lysing end denaturing solutions, wash or rinse solutions, reagents for nucleic acid amplification, electrophoresis and hybridization and labeling and detection reagents. The fluid treatments are unique to each specimen and the detection of target nucleic acid sequences is localized within the carrier device.

Claims

1. A process for handling a single specimen for the analysis of nucleic acid sequences, comprising:

(a) immobilizing said specimen in a device comprising a top cover portion and a bottom housing portion, said top cover portion hingedly moved to a closed position;

said bottom housing portion having an extended area on a first side of said bottom housing portion extending beyond said top cover portion;

wherein said bottom housing portion has edges defining a space, said space comprises a fluid receiving area in the extended area and a thin, flat matrix and specimen holding area;

said fluid receiving area being in fluid communication with said matrix and specimen holding area;

wherein immobilizing the specimen in the matrix and specimen holding area is by means selected from the group consisting of standard fixatives and gel matrix materials;

which matrix and specimen holding area is defined by the top cover portion when said top cover portion is in a closed position;

(b) adding a first treatment fluid to said fluid receiving area wherein the first treatment fluid fills said matrix and specimen holding area forming a liquid film in contact with the top cover portion and the matrix and specimen, said treatment fluids selected from the group consisting of lysing and denaturing solutions, wash and rinse solutions, amplification, hybridization and detection reagents and electrophoresis buffers;

wherein said treatment fluids are added to the device so that the nucleic acids of the immobilized specimen are treated in place on the device;

wherein the top cover portion is in said closed position during amplification;

(c) controlling the temperature of the carrier device for incubating a treatment fluid with the specimen at a desired temperature for a desired time period, said temperature control selected from the group consisting of maintaining and changing the temperature of the device;

(d) adding a volume of a second treatment fluid equal to the volume of the first treatment fluid wherein the first fluid volume exits the matrix and specimen holding area opposite where the fluids are added;

(e) repeating the addition of fluids sufficient to rinse away the prior treatment fluid and bring the next treatment fluid into contact with the specimen in the matrix and specimen holding area;

wherein the detection reagents are used to visualize and enumerate individual locations of the nucleic acids in place within the specimen.

2. A process for specimen handling according to claim 1, wherein step a) comprises mixing the matrix material in a liquid state with the specimen;

wherein the matrix material fills the matrix and specimen holding area with the top cover portion in the closed position and then the matrix material gels embedding the specimen therein;

wherein said gel matrix is dehydrated by heating said device with the device in the open position in such a way that the matrix shrinks to an ultra-thin layer;

wherein said dehydrated gel matrix is rehydrated after dehydration.

3. A process for specimen handling according to claim 1;

wherein the matrix and specimen holding area of said device is divided into sections such that at least one of the sections contains a gel matrix material;

wherein applying current through wires making electrical contact directly with the gel formed in the matrix and specimen holding area and indirectly through fluids in the fluid receiving area;

wherein the electrophoretic transfer of macromolecules from one matrix section to another occurs.

4. A process for specimen handling according to claim 1;

wherein the specimen is suspected of containing at least one specific nucleic acid sequence;

wherein the incubations are repeated with treatment fluids appropriate to enzymatically amplify a specific nuclear acid sequence within the specimen sufficient for detection of the specific nuclear acid sequence; and

wherein the enzymes used are selected from the group consisting of polymerases and ligases.

5. A process for specimen handling for the analysis of according to claim 1;

wherein said detection reagents are selected and include probes containing labels selected from the group consisting of fluorescent, chromogenic and luminescent labels;

said process further comprising microscopically observing the specimen through an optically clear portion of said device.