Patent EP0524808A2 successfully refreshed from Google Patents.
EP0524808A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
In situ PCR preparations, their use, and corresponding thermal cycler equipment
Assignee: F Hoffmann La Roche AG
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Filing: 1992-07-22
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Granted: —
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Expires: 2012-07-22
Abstract
Abstract Improvements to the in situ polymerase chain reaction (PCR), a process of in vitro enzymatic amplification of specific nucleic acid sequences within the cells where they originate, can be achieved by changing the way that the enzymatic reaction is started. Reaction initiation is delayed until the start of PCR thermal cycling, either by withholding a subset of PCR reagents from the cellular preparation until the preparation has been heated to 50 ° to 80 °C, immediately before thermal cycling is begun, or by adding to the PCR reagents a single-stranded DNA binding protein which blocks reaction at temperatures below about 50°C. If the in situ PCR is performed on cellular preparations already attached to a microscope slide, thermal cycling also is facilitated by use of a thermal cycler sample block or compartment designed optimally to hold the microscope slide and any vapor barrier covering the slide.
Inventors
- 👤 Will Bloch
- 👤 Gerard J. Nuovo
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-27 10:06
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6841; B; B01; B01L; B01L7/00; B01L7/52; C12Q1/6844; C12Q1/686
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
21 words
An in situ PCR preparation, comprising fixed cells, a first subset of PCR reagents and optionally a single-stranded DNA binding protein.
Claim 2
Dependent Claim
20 words
An in situ PCR preparation, comprising fixed cells, a complete set of PCR reagents and a single-stranded DNA binding protein.
Claim 3
Dependent Claim
18 words
The preparation of Claim 1 or 2, wherein said fixed cells have been rendered permeable to PCR reagents.
Claim 4
Dependent Claim
18 words
The preparation of any one of Claims 1-3, wherein said fixed cells are attached to a microscope slide.
Claim 5
Dependent Claim
17 words
The preparation of Claim 4, wherein said fixed cells reside within a histochemical section or cytochemical smear.
Claim 6
Dependent Claim
18 words
The preparation of Claim 4 or 5, further comprising a cover slip lying on top of said preparation.
Claim 7
Dependent Claim
20 words
The preparation of any one of Claims 1-6, wherein said single-stranded DNA binding protein is bacteriophage T4 gene 32 protein.
Claim 8
Dependent Claim
22 words
The preparation of any one of Claims 1-6, wherein said single-stranded DNA binding protein is the 19 kilodalton SSB from E. coli.
Claim 9
Dependent Claim
21 words
The preparation of Claim 1, wherein said subset of PCR reagents consists of all PCR reagents except a nucleic acid polymerase.
Claim 10
Dependent Claim
20 words
The preparation of Claims 2 and 4, 5 or 6, comprising a vapor barrier lying on top of said preparation.
Claim 11
Dependent Claim
115 words
A process for in situ PCR amplification of a target nucleic acid sequence, which comprises (a) incubating an in situ PCR preparation comprising fixed cells, a subset of PCR reagents and optionally a single-stranded DNA binding protein at a temperature between about 50° and about 80°C; (b) adding to the preparation of step (a) the PCR reagent subset which complements said first subset; (c) subjecting the preparation of step (b) to thermal cycling sufficient to amplify the target nucleic acid sequence specified by the complete set of PCR reagents and (d) optionally detecting the amplified nucleic acid sequence in a manner which locates it in the individual cells originally containing the target nucleic acid sequence.
Claim 12
Dependent Claim
20 words
The process of Claim 11, wherein said fixed cells are attached to a microscope slide between steps (c) and (d).
Claim 13
Dependent Claim
91 words
The process of Claim 11, wherein (1) said fixed cells are attached to a microscope slide and said preparation is covered with a cover slip before the incubation of step (a), (2) the cover slip is lifted for an interval of between about 5 seconds and about 15 second in order to perform step (b) and then is replaced before performing step (c), (3) a vapor barrier is placed over the cover slip before performing step (c) and (4) the vapor barrier and cover slip are removed before performing step (d).
Claim 14
Dependent Claim
89 words
A process for in situ PCR amplification of a target nucleic acid sequence, which comprises (a) providing an in situ PCR preparation comprising fixed cells, a complete set of PCR reagents and a single-stranded DNA binding protein; (b) subjecting the preparation of step (a) to thermal cycling sufficient to amplify the target nucleic acid sequence specified by the complete set of PCR reagents and (c) optionally detecting the amplified nucleic acid sequence in a manner which locates it in the individual cells originally containing the target nucleic acid sequence.
Claim 15
Dependent Claim
20 words
The process of Claim 14, wherein said fixed cells are attached to a microscope slide between steps (b) and (c).
Claim 16
Dependent Claim
39 words
The process of Claim 14, wherein (1) said fixed cells are attached to a microscope slide, (2) a vapor barrier is placed over said preparation before step (b) and (3) the vapor barrier is removed before performing step (c).
Claim 17
Dependent Claim
18 words
A thermal cycler sample compartment optimized for holding and controlling the temperature of one or more microscope slides.
Claim 18
Dependent Claim
40 words
The thermal cycler sample compartment of Claim 17, comprising a metal block wherein the top surface comprises at least one horizontal flat area with dimensions of at least about 17 mm x 16 mm sufficient to hold a microscope slide.
Claim 19
Dependent Claim
25 words
The sample block of Claim 18, further comprising at least one row of wells shaped to hold conical microcentrifuge tubes of approximately 0.5 ml capacity.
Claim 20
Dependent Claim
43 words
The thermal cycler sample compartment of Claim 17, comprising (a) a chamber designed to contain and direct the flow of a moving heat transfer fluid and (b) a device within the chamber which holds microscope slides firmly in the moving heat transfer fluid.
Claim 21
Dependent Claim
36 words
A plastic envelope, substantially impermeant to liquid water and water vapor at temperatures between about 25° and about 100°C, with inner dimensions of length, width and height approximately the same as those of a microscope slide.
References Cited (Backward Citations)
No citation records indexed.
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