Device for processing biological specimens for analysis of nucleic acids

• US 5,451,500 A
• Filed: 03/03/1992
• Issued: 09/19/1995
• 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): Houtteman, Scott W.
• Application Number:
  US07/836,348
• Time in Patent Office: 1,295 Days
• Field of Search: 435/6, 435/290, 435/294, 435/289, 935/87
• US Class Current: 435/6.11
• 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 system is provided for the automated detection of target nucleic acid sequences in which multiple biological samples are individually incorporated into matrices within carriers in a two-dimensional format. The matrix carrier is an inexpensive, disposable unit that represents a closed system separating the specimens. The system integrates sample preparation within the matrix and facilitates biochemical reactions, addition of reagents and washes, removal of waste fluids, temperature control and the automated processing thereof. The carriers are processed in stepwise treatments that expose, amplify, and detect the presence or absence of specific genetic entities in each sample. The nucleic acids or other desired biological components held within the matrices are treated by one or more of the techniques such as amplification, electrophoresis, analyte-receptor binding or hybridization as selected for the desired analysis. Different types of carriers are used for different kinds of diagnostic tests or test panels. Different primer and polymerase molecules may be used to replicate the target sequences of nucleic acids in the sample. The system also includes a heating and a passive cooling system that will provide rapid thermal cycling in a two-dimensional format for denaturation and amplification protocols so requiring them, or maintain constant temperatures for isothermal amplification and hybridization protocols. The system is adaptable to a variety of non-isotopic labels. The two-dimensional format lends itself to digitizing signals in a two-dimensional array for image analysis. Microscopic interpretation is possible with or without image analysis.

Claims

1. An apparatus for automated liquid delivery to and precise temperature control of samples immobilized on a surface of a carrier device for the detection of genetic material comprising:

(a) a reaction chamber for situating the samples, said samples individually immobilized in a thin, flat carrier device in said apparatus during treatments;

said carrier device comprising a top piece and a bottom piece, said bottom piece having a specimen holding area, said top piece having a closed position;

said top piece and said bottom piece hinged together along a first side of said bottom piece, said top piece having a first area that extends beyond said first side, wherein the top piece may be caused to hingedly move away from the bottom piece and upward from the closed position to an open position; and

said bottom piece having an overlap area on a second side of said bottom piece, said overlap area extending beyond said top piece, said overlap area having a fluid receiving area;

said reaction chamber containing a tray rack for holding a plurality of carrier devices;

(b) reservoirs for treatment liquids;

(c) treatment liquid transfer lines, said reservoirs connected to the reaction chamber by means of said treatment liquid transfer lines;

(d) means to control the timing and volume of treatment liquids delivered to samples through liquid transfer lines;

said carriers situated in said tray rack in a generally horizontal position so that liquids are dispensed sequentially into an end of each carrier and flow over said immobilized sample and the liquids exit the carrier at the end of the carrier opposite the end of the carrier where liquids are dispensed;

(e) a pump to cause the treatment liquids to move through the treatment liquid transfer lines to the samples in the reaction chamber,

(f) means to regulate and rapidly change temperature of the samples in said flat carrier devices in the reaction chamber; and

(g) means to contain used treatment liquids away from the samples;

wherein liquid delivery and precise temperature control are controlled by a programmable central microprocessor.

2. An apparatus according to, claim 1, further comprising a thin, flat matrix in said carrier device selected from a group consisting of agarose, polyacrylamide, and mixtures thereof, with means to supply electric current to said matrix situated in each said carrier device positioned in said tray rack in a way that each said matrix completes an electrical circuit for electrophoresis.

3. A method for studying genetic material of a sample in comprising the steps of:

(a) providing a thin, flat semi-solid gel matrix containing the sample in an apparatus comprising;

(i) a reaction chamber containing a tray rack for holding a plurality of thin, flat samples;

(ii) means to control the timing and volume of sequential treatment fluids delivered to each said sample;

(iii) means to regulate and rapidly change temperature of each said

(b) treating the samples immobilized in said gel matrix with treatment liquids in an amount effective to remove interfering, non-genetic material from the sample;

(c) dehydrating the matrix to decrease volume of said matrix;

(d) saturating the matrix with a first reagent;

(e) denaturing the genetic material;

(f) rapidly adjusting temperature for nucleic acid strand melting and annealing;

(g) enzymatically amplifying the genetic material to be identified;

wherein said amplifying is selected from a group consisting of polymerase and ligase techniques, and wherein specific, native nucleic acid sequences are used as templates repeatedly;

(h) repeating steps (e) through (g) until a specific nucleic acid target is amplified sufficiently to be detected; and

(i) rinsing away reagents and repeating steps (d) through (f) with appropriate reagents to detect specific genetic material within the matrix;

wherein steps (b) through (i) are done within the said apparatus.

4. A method for studying genetic material in a sample according to claim 3, further comprising spreading nucleic acid fragments in the said gel matrix by subjecting them to electrophoresis before step (e) of claim 3 or after step (h) of claim 3.

5. A process of specimen handling for analysis of a particular nucleic acid sequence of a specimen, comprising:

(a) immobilizing a flat thin specimen in a specimen holding area in a carrier device, said carrier device comprising;

a top piece and a bottom piece, said bottom piece having a specimen holding area, said top piece having a closed position;

said top piece and said bottom piece hinged together along a first side of said bottom piece, said top piece having a first area that extends beyond said first side, wherein the top piece may be caused to hingedly move away from the bottom piece and upward from the closed position to an open position; and

said bottom piece having an overlap area on a second side of said bottom piece, said overlap area extending beyond said top piece, said overlap area having a fluid receiving area;

said carrier device in an apparatus comprising;

(i) a reaction chamber containing a tray rack for holding a plurality of thin, flat specimens;

(ii) means to control the timing and volume of sequential treatment fluids delivered to each of said specimens;

(iii) means to regulate and rapidly change temperature of each of said specimens;

(b) providing a zone for fluid entry to the specimen, contact of the fluid with the specimen and exit of waste fluid from the specimen;

(c) alternately heating and cooling the carrier device to regulate specimen temperatures needed for nucleic acid strand denaturation, annealing of specific primers, and enzymatic amplification; and

(d) processing the specimen by utilizing one or more of the following methods;

(i) amplifying in situ by primer extensions using a polymerase, wherein each specific, native nucleic acid target is repeatedly used as a template;

(ii) amplifying in situ by primer extensions using a polymerase, wherein each specific, native nucleic acid target and said primer extensions are repeatedly used as templates; and

(iii) amplifying in situ by binding oligonucleotide sequences to their specific target sequences so that the oligonucleotides are directly adjacent and repeatedly ligating said adjacent oligonucleotides;

wherein all processing of the specimen is preformed in the carrier device and wherein the presence and position of localized original nucleic acids targets in the specimen are distinguished and enumerated microscopically in situ.