Rolling circle amplification detection of RNA and DNA
AssigneeThe Regents of the University of California
InventorsAllen T. Christian, Melissa S. Pattee, Cristina M. Attix, James D. Tucker
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Abstract
Rolling circle amplification (RCA) has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate RCA in solution and in situ to detect gene copy number and single base mutations. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.
Claims35
A method of rolling circle amplification of DNA comprising:
- a) providing DNA;
- b) digesting said DNA with an endonuclease to form nicked DNA;
- c) digesting said nicked DNA with an exonuclease to prepare exonuclease-treated DNA for rolling circle amplification;
- and
- d) performing rolling circle amplification on said exonuclease-treated DNA.
A method of preparing DNA for rolling circle amplification in situ, comprising:
- a) fixing cells on a surface wherein said cells comprise DNA;
- b) digesting said DNA on said surface with an endonuclease to form nicked DNA;
- and
- c) digesting said nicked DNA with an exonuclease to prepare said DNA for rolling circle amplification.
A method of performing rolling circle amplification in situ, comprising:
- a) fixing cells on a surface wherein said cells comprise DNA;
- b) digesting said DNA on said surface with an endonuclease to form nicked DNA;
- c) digesting said nicked DNA with an exonuclease to form target DNA;
- d) ligating a padlock oligonucleotide probe to said target DNA to form ligated DNA;
- and
- e) performing rolling circle amplification in situ on said ligated DNA.
A method of performing rolling circle amplification in situ, comprising:
- a) providing cells embedded in paraffin wherein said cells comprise DNA;
- b) digesting said DNA in said paraffin with an endonuclease to form nicked DNA;
- c) digesting said nicked DNA with an exonuclease to form target DNA;
- d) ligating a padlock oligonucleotide to said target DNA to form ligated DNA;
- and
- e) performing rolling circle amplification in situ on said ligated DNA.
A method of detecting RNA in situ, comprising;
- a) fixing cells on a surface with ethanol wherein said cells comprise RNA;
- and
- b) performing rolling circle amplification in situ on said RNA to detect said RNA in situ.
A method for performing rolling circle amplification in situ to detect RNA, comprising:
- a) centrifuging cells onto a surface;
- b) fixing said cells on said surface with ethanol;
- c) hybridizing a padlock oligonucleotide probe to said RNA to form a DNA-RNA hybrid;
- d) ligating said oligonucleotide probe to said DNA of said DNA-RNA hybrid to form ligated DNA;
- e) performing,rolling circle amplification on said ligated DNA to form amplified DNA;
- and
- f) detecting said amplified RNA by determining the levels of said amplified DNA.
Dependencies are inferred from the claim text.