Methods for Nucleic Acid Mapping and Identification of Fine Structural Variations in Nucleic Acids
First Claim
1. An in vitro, extracellular method of juxtaposing sequence tags (GVTs) where two constituent members of a tag pair (GVT-pair) are unique positional markers of a defined separation distance in a plurality of target nucleic acid molecules, the method comprising:
- fragmenting the target nucleic acid molecule to form target DNA insert;
ligating a DNA adaptor having one or more restriction endonuclease recognition sites to both ends of a fragmented target DNA insert and the ligation of the adaptor-ligated target DNA to a DNA backbone to create a circular molecule;
digesting the adaptor using a type IIS, type IIG, or type III restriction endonuclease at the recognition site to cleave the target DNA insert at a defined distance from each end thereof to create two sequence tags (GVTs) comprising terminal sequences of the target DNA insert that are attached to the linear DNA backbone;
recircularizing the linear DNA backbone with the attached GVTs to obtain a circular DNA molecule including a GVT pair having two juxtaposed GVTs;
isolation of juxtaposed GVT-pair by nucleic acid amplification.
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Abstract
An in vitro, extracellular method of juxtaposing sequence tags (GVTs) where two constituent members of a tag pair (GVT-pair) are unique positional markers of a defined separation distance and/or are markers of nucleic acid positions that demarcate adjacent cleavage sites for one or more different restriction endonucleases along the length of a plurality of target nucleic acid molecules, the method comprising: Fragmenting the target nucleic acid molecule to form target DNA insert; ligating a DNA adaptor having one or more restriction endonuclease recognition sites to both ends of a fragmented target DNA insert and the ligation of the adaptor-ligated target DNA insert to a DNA backbone to create a circular molecule; digesting the adaptor using a restriction endonuclease at the recognition site to cleave the target DNA insert at a defined distance from each end thereof to create two sequence tags (GVTs) comprising terminal sequences of the target DNA insert that are attached to the linear DNA backbone; and recircularizing the linear DNA backbone with the attached GVTs to obtain a circular DNA molecule including a GVT pair having two juxtaposed GVTs; GVT-pair DNA is recovered by nucleic acid amplification.
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Citations
40 Claims
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1. An in vitro, extracellular method of juxtaposing sequence tags (GVTs) where two constituent members of a tag pair (GVT-pair) are unique positional markers of a defined separation distance in a plurality of target nucleic acid molecules, the method comprising:
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fragmenting the target nucleic acid molecule to form target DNA insert; ligating a DNA adaptor having one or more restriction endonuclease recognition sites to both ends of a fragmented target DNA insert and the ligation of the adaptor-ligated target DNA to a DNA backbone to create a circular molecule; digesting the adaptor using a type IIS, type IIG, or type III restriction endonuclease at the recognition site to cleave the target DNA insert at a defined distance from each end thereof to create two sequence tags (GVTs) comprising terminal sequences of the target DNA insert that are attached to the linear DNA backbone; recircularizing the linear DNA backbone with the attached GVTs to obtain a circular DNA molecule including a GVT pair having two juxtaposed GVTs; isolation of juxtaposed GVT-pair by nucleic acid amplification. - View Dependent Claims (3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39)
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2. An in vitro, extracellular method of juxtaposing sequence tags (GVTs) where the two constituent members of a tag pair are unique positional markers of two adjacent and cleavable restriction endonuclease sites of one or more restriction endonucleases along the length of a plurality of target nucleic acid molecules, the method comprising:
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fragmenting the target nucleic acid molecule DNA by digestion with one or more restriction endonucleases to form target DNA insert; ligating a digested target DNA insert to a linear DNA backbone to create a circular DNA molecule whereby the target DNA insert is flanked by a pair of recognition sites for a type IIS, type IIG, or type III restriction endonuclease; digesting the target DNA insert using a type IIS, type IIG, or type III restriction endonuclease restriction at the recognition site to cleave the target DNA insert at a defined distance from each end of the target DNA insert to create two sequence tags (GVTs) comprising terminal sequences of the target DNA insert that are attached to the linear DNA backbone; and recircularizing the linear DNA backbone with the attached GVTs to form a circular molecule bearing a GVT-pair comprising two juxtaposed GVTs; isolation of juxtaposed GVT-pair by nucleic acid amplification. - View Dependent Claims (4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 30, 32, 34, 36, 38, 40)
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28. The method of claim L, wherein the type III restriction endonuclease is used to create the GVT is Pst II.
Specification