Fluorescent in situ rt-pcr
First Claim
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1. A method for in situ amplification of a target nucleic acid in a selected cell comprising:
- a) contacting a fixed, permeabilized cell with a restriction endonuclease composition comprising at least one restriction endonuclease to produce a restriction digest;
b) contacting said cell with a DNase to produce a DNase digested cell;
c) incubating said cell with a reverse transcriptase (RT) cocktail comprising an RT enzyme and a RT primer specific for said target nucleic acid to produce a cDNA; and
d) amplifying said cDNA using a PCR reaction in the presence of forward and reverse PCR primers specific for said target nucleic acid wherein at least one of said PCR primers is labeled.
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Abstract
The present invention describes an in situ reverse transcriptase PCR method in which the background fluorescence is greatly reduced as compared to traditional in situ PCR.
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Citations
42 Claims
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1. A method for in situ amplification of a target nucleic acid in a selected cell comprising:
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a) contacting a fixed, permeabilized cell with a restriction endonuclease composition comprising at least one restriction endonuclease to produce a restriction digest;
b) contacting said cell with a DNase to produce a DNase digested cell;
c) incubating said cell with a reverse transcriptase (RT) cocktail comprising an RT enzyme and a RT primer specific for said target nucleic acid to produce a cDNA; and
d) amplifying said cDNA using a PCR reaction in the presence of forward and reverse PCR primers specific for said target nucleic acid wherein at least one of said PCR primers is labeled. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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- 2. The method of claim 2, wherein at least one of said PCR primers is labeled with a radioactive label, an immunocytochemical label or a fluorescent label to facilitate detection of said amplified cDNA.
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30. A method for in situ amplification of a target nucleic acid in a selected cell comprising:
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a) contacting a fixed, permeabilized cell with a restriction endonuclease composition comprising Sau 96I to produce a restriction digest;
d) contacting said restriction digest with a DNase to produce a DNase digested cell;
e) incubating said DNase digested cell with a reverse transcriptase (RT) cocktail contacting an RT enzyme and a RT primer specific for said target nucleic acid to produce a cDNA; and
f) amplifying said cDNA using a polymerase chain reaction (PCR) reaction in the presence of forward and reverse PCR primers specific for said target nucleic acid wherein at least one of said PCR primers is labeled to facilitate detection wherein said contacting of said cell with a said restriction endonuclease composition produces an increased signal for detection as compared to an in situ RT PCR reaction conducted in the absence of said restriction endonuclease composition. - View Dependent Claims (31)
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32. A method for improving the signal from in situ RT PCR reaction comprising subjecting the target cell to a restriction endonuclease reaction and DNase digestion.
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33. A method for improving the signal from in situ RT PCR reaction comprising conducting PCR reaction in the presence of at least one PCR primer labeled with a fluorescent label that fluoresces in the far red range of fluorescence range.
- 34. A kit for use in in situ RT PCR, said kit comprising discrete containers of a first restriction endonuclease, a second restriction endonuclease, and an RNase free DNase.
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41. In a method for carrying out an in situ RT PCR reaction on a fixed cell, the improvement comprising the steps of:
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(a) contacting the fixed cell with a restriction endonuclease composition to produce a restriction digest, and (b) contacting the fixed cell with a DNase composition to produce a DNase digest.
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42. In a method for carrying out an in situ RT PCR reaction on a fixed cell, the improvement comprising the step of conducting PCR reaction in the presence of at least one PCR primer labeled with a fluorescent label that fluoresces in the far red range of fluorescence range.
Specification