Method for studying nucleic acids within immobilized specimens

• US 5,382,511 A
• Filed: 08/24/1992
• Issued: 01/17/1995
• Est. Priority Date: 08/02/1988
• 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): Marschel, Ardin H.
• Application Number:
  US07/935,637
• Time in Patent Office: 876 Days
• Field of Search: 435/6, 435/7.1, 435/91, 435/7.2, 435/810, 435/287, 435/91.1, 435/291, 435/91.2, 435/5, 436/501, 436/810, 536/22.1-24.1, 935/78, 935/88
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: G01N 1/31 Apparatus therefor

Abstract

An apparatus for the automated detection of target nucleic acid sequences in which multiple biological samples are individually incorporated into a matrix and treated in a stepwise fashion to expose, amplify, detect and enumerate the presence of original, target nucleotide sequences in each sample. The samples are held together with other samples in a chamber connected by means of a fluid flow conduit to which the matrices make connection via numerous jet-spray ports for the injection of various fluids from a plurality of reservoirs used to process the samples. Solvents and reagents are used to degrade and wash cellular structures and contaminants away from the nucleic acids. Primer and polymerase molecules are used to replicate the target sequences of nucleic acids in the sample. Labeled DNA probe molecules serve to hybridize to the amplified target sequences for detection. Appropriate buffers allow these manipulations on the nucleic acid molecules in the samples. The system also includes a pump to draw the fluids from the reservoirs and force them through the jet spray manifolds into the main chamber and valves under the control of a microprocessor. The system also includes fans and heating elements for regulating temperature and humidity. Different nucleic acid targets in different biological specimens are accommodated by regulating the quantity, duration, pH, temperature and pressure of fluids flowing from the reservoirs.

Claims

1. A method for studying genetic material of a sample in situ, comprising the steps of:

(a) preparing a thin agarose matrix containing the sample in which the genetic material of the sample is anchored in separate positions in the matrix; and

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

said treatment utilizing an apparatus comprising;

(i) a reaction chamber for situating the samples;

(ii) reservoirs for treatment liquids;

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

(iv) means to control the addition to the reaction chamber of the treatment liquids in the reservoirs;

(v) means to regulate air flow and temperature in the reaction chamber;

(vi) a pump to cause the treatment liquids to move in the treatment liquid transfer lines; and

(vii) means to remove treatment liquids from the reaction chamber;

(c) amplifying target genetic material in the samples, wherein amplifying the genetic material consists essentially of utilizing repetitive enzymatic techniques selected from a group consisting of polymerase techniques to synthesize new DNA complementary to nucleic acid targets and ligase techniques to join together oligonucleotide sequences bound to their complementary nucleic acid targets when said oligonucleotide sequences bound to their nucleic acid targets are directly adjacent;

(d) determining and enumerating the presence of nucleic acid targets;

wherein all sample treatment steps are done in a generally horizontal matrix.

2. A method for studying genetic material in a sample according to claim 1, wherein the sample is selected from a group consisting of cells and viruses and mixtures of cells and viruses, wherein nucleic acid targets are used to distinguish certain cells or viruses.

3. A method for studying genetic material in a sample according to claim 1, wherein the sample comprises tissue specimens, wherein nucleic acid targets are used to distinguish and enumerate certain cells in tissue from other cells.

4. A method for studying genetic material of a sample according to claim 1, wherein the matrix comprises an agarose gel prepared in such a way so that the matrix can be subjected to electrophoresis, and wherein the sample is subjected to electrophoresis in the agarose gel after step (c).

5. A method for studying genetic material in a sample according to claim 1, wherein a plurality of samples is placed in the apparatus and treated simultaneously, wherein each of said samples is individually embedded in a separate matrix section.

6. A method for studying genetic material of a sample according to claim 1, further comprising

hybridizing a labeled nucleic acid probe to the genetic material.

7. A method for studying genetic material in a sample according to claim 1, wherein the matrix treatment steps comprise:

(a) lysing the non-genetic material in the sample;

(b) washing the matrix;

(c) denaturing the genetic material;

(d) neutralizing the genetic material

(e) dehydrating the matrix; and

the amplification step comprises;

(f) rehydrating the matrix with solution containing nucleic acid amplification treatment liquids;

(g) adjusting temperatures for amplification;

(h) partially dehydrating the matrix during nucleic acid denaturation; and

(i) repeating steps (f) through (h) until the nucleic acid is amplified sufficiently to be detectable by labeled probes.

8. A method for studying genetic material of a sample according to claim 1, wherein the matrix is prepared as a gel using a standard method for preparing gels for electrophoresis prior to step (a), and wherein the gel is treated with treatment liquids following electrophoresis of the genetic material in the gel.

11. A method for studying genetic material in a sample according to claim 8, wherein the sample comprises tissue specimens, wherein nucleic acid targets are used to distinguish and enumerate certain cells in tissue from other cells.

9. A method for studying genetic material of a sample in situ, comprising the steps of:

(a) fixing the sample containing the genetic material on a thin, flat support;

(b) treating the sample with treatment liquids in an amount effective to remove interfering material from the genetic material of the sample;

(c) amplifying the genetic material to be identified by synthesizing new DNA or RNA utilizing one or more of the following methods;

(i) priming a specific sequence and extending synthesis with a polymerase, wherein said specific sequence is repeatedly used as a template;

(ii) priming and extending synthesis of a specific sequence with two opposing primers and a polymerase to form primer extensions, wherein said specific sequence and the resulting primer extensions are repeatedly used as templates;

(iii) binding oligonucleotide sequences to specific target sequences so that the oligonucleotide sequences are directly adjacent to each other and repeatedly ligating said adjacent oligonucleotide sequences; and

(d) detecting said localized amplified genetic material by means selected from a group comprising the incorporation of labeled nucleotides and hybridizing labeled oligonucleotides to the amplified genetic material;

wherein all the steps are done with the sample containing the genetic material to be identified fixed on said thin, flat support.

10. A method for studying genetic material in a sample according to claim 9, wherein the sample is selected from a group consisting of cells and viruses and mixtures of cells and viruses, wherein nucleic acid targets are used to distinguish and enumerate certain cells or viruses.

12. A method for studying the genetic material of a sample according to claim 9 wherein at least one specific mRNA sequence contained in the genetic material of a biological specimen in situ is amplified, and said method further comprises firstly synthesizing cDNA within said specimen by repeated, primer extensions of a specific mRNA sequence in the presence of reverse transcriptase, and denaturing the cDNA:

RNA hybrid molecules and polymerizing second strand DNA within said specimen by primer extensions on the cDNA templates in the presence of polymerase and primers that are complementary to the cDNA, wherein said method enables certain cells in the specimen containing said specific mRNA sequence to be enumerated and distinguished.

13. A method for studying genetic material in a sample wherein the sample comprises tissue sections in which treatment steps are done in a thin, flat, generally horizontal, format;

comprising the steps of;

(a) preparing the tissue sections on microscope slides by standard methods;

(b) providing an apparatus for the treatments comprising;

(i) a reaction chamber for situating the samples;

(ii) reservoirs for treatment liquids;

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

(iv) means to control the addition to the reaction chamber of the treatment liquids in the reservoirs;

(v) means to regulate air flow and temperature in the reaction chamber;

(vi) a pump to cause the treatment liquids to move in the treatment liquid transfer lines; and

(vii) means to remove treatment liquids from the reaction chamber;

(c) exposing and denaturing the nucleic acids in the samples;

(d) repeatedly amplifying the genetic materials;

(e) hybridizing a labled nucleic acid probe to the gentic material; and

(f) determining the presence of hybridized labled probe;

wherein the steps (c) through (f) are performed on the microscope slides in said apparatus before microscopic analysis.

14. A method for studying genetic material in a sample in which the following treatment steps are done in a thin, flat, generally horizontal, agarose matrix;

comprising the steps of;

(a) preparing the sample in said matrix;

(b) denaturing the nucleic acids in the samples;

(c) repeatedly amplifying the genetic material to be identified;

(d) hybridizing a labeled nucleic acid probe to the genetic material; and

(e) determining the presence of hybridized labeled probe.

15. A method for studying genetic material in samples according to claim 14 wherein treatment steps a-e utilize an apparatus comprising:

(a) a reaction chamber for situating the samples;

(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 addition to the reaction chamber of the treatment liquids in the reservoirs;

(e) means to regulate air flow and temperature in the reaction chamber;

(f) a pump to cause the treatment liquids to move in the treatment liquid transfer lines; and

(g) means to remove treatment liquids from the reaction chamber.

16. A method for studying genetic material of a sample in situ, comprising the steps of:

(a) preparing a thin, flat, semi-solid matrix containing the sample;

said matrix comprising a material which is pourable as a liquid and is capable of immobilizing the sample when cooled to form a gel, said gel having a pore size which allows entry and exit of treatment liquids while fixing the positional orientation of nucleic acid targets within the sample;

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

(c) repeatedly amplifying the genetic material to be identified in said matrix; and

(d) utilizing the amplified genetic material for detection of specific genetic sequences.