Vaccinia topoisomerases I-based assays for detection of specific DNA breaks
First Claim
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1. A method of detecting apoptotic cells in a cellular sample, comprising the steps of obtaining a cellular sample;
- contacting the cellular sample with a solution comprising a nucleic acid molecule and a topoisomerase I enzyme, the nucleic acid molecule being cleavable by the topoisomerase I enzyme and ligatable to a 5′
OH; and
detecting the nucleic acid molecule ligated to a 5′
OH of the DNA of the cellular sample, wherein the detection of the nucleic acid molecule ligated to a 5′
OH of the DNA of the cellular sample correlates to the presence of apoptotic cells.
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Abstract
Several types of DNA cuts are used as markers of apoptosis for detection of apoptotic cells in situ. The present invention includes a method to detect DNase II type DNA damage, bearing 5′OH using vaccinia topoisomerase I. The present invention also includes a method combining a ligase-based method to detect DNase I type DNA damage and the topoisomerase based method, resulting in simultaneous detection of two specific types of DNA damage in situ.
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Citations
50 Claims
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1. A method of detecting apoptotic cells in a cellular sample, comprising the steps of obtaining a cellular sample;
- contacting the cellular sample with a solution comprising a nucleic acid molecule and a topoisomerase I enzyme, the nucleic acid molecule being cleavable by the topoisomerase I enzyme and ligatable to a 5′
OH; and
detecting the nucleic acid molecule ligated to a 5′
OH of the DNA of the cellular sample, wherein the detection of the nucleic acid molecule ligated to a 5′
OH of the DNA of the cellular sample correlates to the presence of apoptotic cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
- contacting the cellular sample with a solution comprising a nucleic acid molecule and a topoisomerase I enzyme, the nucleic acid molecule being cleavable by the topoisomerase I enzyme and ligatable to a 5′
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27. A method of detecting apoptotic cells in a cellular sample comprising the steps of:
- isolating DNA from a cellular sample;
contacting the DNA with a solution comprising a nucleic acid molecule and a topoisomerase I enzyme, the nucleic acid molecule being cleavable by the topoisomerase I enzyme and ligatable to a 5′
OH; and
detecting the nucleic acid molecule ligated to a 5′
OH of the DNA, wherein the detection of the nucleic acid molecule ligated to a 5′
OH of the DNA correlates to the presence of apoptotic cells. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)
- isolating DNA from a cellular sample;
Specification