US5538871A
Expired / Lapsed
US
⏱️ 0.0 years remaining
In situ polymerase chain reaction
Assignee: Roche Molecular Systems Inc
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Filing: 1995-02-17
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Granted: 1996-07-23
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Expires: 2015-02-17
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 DEG C. to 80 DEG 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 DEG 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
- 👤 Gerard J. Nuovo
- 👤 Will Bloch
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 12:02
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: 26
Claim 1
Independent Claim
191 words
A method for in situ PCR amplification of target nucleic acid wherein said amplified nucleic acid is spatially confined to individual cells originally containing said target nucleic acid, said method comprising: (a) fixing cells thought to contain said target nucleic acid in a fixative selected from the group consisting of formalin, formaldehyde, paraformaldehyde and glutaraldehyde; (b) contacting said fixed cells with a proteinase; (c) adding a first subset of PCR reagents to said fixed cells, said first subset of PCR reagents comprising a portion of the PCR reagents necessary to allow PCR to proceed; (d) incubating in a moist environment said fixed cells and said first subset of PCR reagents at a temperature between 50° C. and about 80° C.; (e) adding a second subset of PCR reagents to said fixed cells, said second subset of PCR reagents comprising the PCR reagents which are missing from said first subset of PCR reagents and which are necessary to allow PCR to proceed; and (f) subjecting said fixed cells and said first and said second subsets of PCR reagents in a moist environment to thermal cycling sufficient to amplify said target nucleic acid.
Claim 2
Independent Claim
191 words
A method for in situ PCR amplification of target nucleic acid wherein said amplified nucleic acid is spatially confined to individual cells originally containing said target nucleic acid, said method comprising: (a) fixing cells thought to contain said target nucleic acid in a fixative selected from the group consisting of formalin, formaldehyde, paraformaldehyde and glutaraldehyde; (b) contacting said fixed cells with a proteinase; (c) adding a first subset of PCR reagents to said fixed cells, said first subset of PCR reagents comprising a portion of the PCR reagents necessary to allow PCR to proceed; (d) incubating in a moist environment said fixed cells and said first subset of PCR reagents at a temperature between 50° C. and about 80° C.; (e) adding a second subset of PCR reagents to said fixed cells, said second subset of PCR reagents comprising the PCR reagents which are missing from said first subset of PCR reagents and which are necessary to allow PCR to proceed; and (f) subjecting said fixed cells and said first and said second subsets of PCR reagents in a moist environment to thermal cycling sufficient to amplify said target nucleic acid.
Claim 3
Dependent Claim
13 words
A method in accordance with claim 1 wherein said cells are animal cells.
Claim 4
Dependent Claim
12 words
A method in accordance with claim 1 wherein said fixative is formalin.
Claim 5
Dependent Claim
13 words
A method in accordance with claim 3 wherein said formalin is buffered formalin.
Claim 6
Dependent Claim
22 words
A method in accordance with claim 4 wherein said buffered formalin is 10% formalin in 0.1 M sodium phosphate buffer, pH 7.0.
Claim 7
Dependent Claim
25 words
A method in accordance with claim 1 wherein said fixed cells re, side within a histochemical section or cytochemical smear attached to a microscope slide.
Claim 8
Dependent Claim
21 words
A method in accordance with claim 1 wherein prior to step (d) said fixed cells are covered with a cover slip.
Claim 9
Dependent Claim
21 words
A method in accordance with claim 1 wherein said fixed cells are attached to a microscope slide prior to step (c).
Claim 10
Dependent Claim
24 words
A method in accordance with claim 1 wherein said first subset of PCR reagents consists of all PCR reagents except a nucleic acid polymerase.
Claim 11
Dependent Claim
32 words
A method in accordance with claim 1 further comprising: (g) detecting said amplified nucleic acid in a manner which locates it in the individual cells originally containing said target nucleic acid sequence.
Claim 12
Dependent Claim
22 words
A method in accordance with claim 10 wherein said fixed cells are attached to a microscope slide between steps (f) and (g).
Claim 13
Independent Claim
101 words
A method for in situ PCR amplification of target nucleic acid wherein said amplified nucleic acid is spatially confined to individual cells originally containing said target nucleic acid, said method comprising: (a) fixing cells thought to contain said target nucleic acid in a fixative selected from the group consisting of formalin, formaldehyde, paraformaldehyde and glutaraldehyde; (b) contacting said fixed cells with a proteinase; (c) adding a complete set of PCR reagents and a single-stranded DNA binding protein to said fixed cells; and (d) subjecting said fixed cells in a moist environment to thermal cycling sufficient to amplify said target nucleic acid.
Claim 14
Independent Claim
101 words
A method for in situ PCR amplification of target nucleic acid wherein said amplified nucleic acid is spatially confined to individual cells originally containing said target nucleic acid, said method comprising: (a) fixing cells thought to contain said target nucleic acid in a fixative selected from the group consisting of formalin, formaldehyde, paraformaldehyde and glutaraldehyde; (b) contacting said fixed cells with a proteinase; (c) adding a complete set of PCR reagents and a single-stranded DNA binding protein to said fixed cells; and (d) subjecting said fixed cells in a moist environment to thermal cycling sufficient to amplify said target nucleic acid.
Claim 15
Dependent Claim
13 words
A method in accordance with claim 12 wherein said cells are animal cells.
Claim 16
Dependent Claim
12 words
A method in accordance with claim 12 wherein said fixative is formalin.
Claim 17
Dependent Claim
13 words
A method in accordance with claim 14 wherein said formalin is buffered formalin.
Claim 18
Dependent Claim
22 words
A method in accordance with claim 15 wherein said buffered formalin is 10% formalin in 0.1 M sodium phosphate buffer, pH 7.0.
Claim 19
Dependent Claim
20 words
A method in accordance with claim 11 wherein said singles stranded DNA binding protein is bacteriophage T4 gene 32 protein.
Claim 20
Dependent Claim
21 words
A method in accordance with claim 12 wherein said single-stranded DNA binding protein is the 19 kilodalton SSB from E. coli.
Claim 21
Dependent Claim
24 words
A method in accordance with claim 12 wherein said fixed cells reside within a histochemical section or cytochemical smear attached to a microscope slide.
Claim 22
Dependent Claim
21 words
A method in accordance with claim 12 wherein prior to step (c) said fixed cells are covered with a cover slip.
Claim 23
Dependent Claim
21 words
A method in accordance with claim 12 wherein prior to step (c) said fixed cells are covered with a vapor barrier.
Claim 24
Dependent Claim
32 words
A method in accordance with claim 12 further comprising: (e) detecting said amplified nucleic acid in a manner which locates it in the individual cells originally containing said target nucleic acid sequence.
Claim 25
Dependent Claim
21 words
A method in accordance with claim 22 wherein said fixed cells are attached to a microscope slide prior to step (c).
Claim 26
Dependent Claim
22 words
A method in accordance with claim 22 wherein said fixed cells are attached to a microscope slide between steps (d) and (e).
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-07-23 | 2000-01-22 | 2000-07-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-07-23 | 2004-01-22 | 2004-07-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-07-23 | 2008-01-22 | 2008-07-23 | $7700.00 | Window Closed / Lapsed |