Methods for nucleic acid mapping and identification of fine-structural-variations in nucleic acids
First Claim
1. A method for producing juxtaposing sequence tags (GVTs) for determining a fine-structural-variation of a subject nucleic acid molecule, where two constituent members of a sequence tag pair (GVT-pair) located along the length of a population of target nucleic acid molecules are positional markers of a defined separation distance, and the fine-structural-variation is selected from insertion, deletion, duplication, inversion, translocation, or nucleic acid sequence rearrangement;
- wherein the method comprises;
fragmenting the subject nucleic acid molecule to form a target DNA insert;
ligating the target DNA insert to a linear DNA backbone at terminal cloning sites of the target DNA insert, leading to the creation of a circular molecule comprising the target DNA insert;
digesting the target DNA insert within the circular molecule using at least one endonuclease cleaving the target DNA insert at a distance from each of the insert'"'"'s terminal cloning sites and thereby creating a linear molecule comprising two sequence tags (GVTs) comprising the terminal sequences of the target DNA insert, one of the two GVTs attached to each end of the undigested DNA backbone;
recircularizing the linear DNA backbone with the attached GVTs to create a circular DNA molecule thereby creating a GVT-pair comprising two juxtaposed GVTs that are the same relative orientation as the target DNA insert;
isolating the created GVT-pair by nucleic acid amplification from primer sites on the DNA backbone or by digestion with endonuclease at sites that are on the DNA backbone and are flanking the created GVT-pair;
corresponding the GVT-pair to a reference nucleic acid molecule, wherein each GVT corresponds to a segment of the reference nucleic acid molecule;
determining a structural information of interest of the GVT-pair on the subject nucleic acid molecule, wherein the structural information of interest of the GVT-pair on the subject nucleic acid molecule is selected from location of the GVT-pair on the subject nucleic acid molecule, separation distance between the GVT-pair on the subject nucleic acid molecule, or orientation between the GVT-pair on the subject nucleic acid molecule;
determining a corresponding structural information of interest of the segments on the reference nucleic acid molecule, wherein the corresponding structural information of interest of the segments on the reference nucleic acid molecule corresponds to the structural information of interest of the GVT-pair on the subject nucleic acid molecule, and the corresponding structural information of interest of the segments on the reference nucleic acid molecule is selected from location of the segments on the reference nucleic acid molecule, separation distance between the segments on the reference nucleic acid molecule, or orientation between the segments on the reference nucleic acid molecule; and
determining whether the subject nucleic acid molecule has a fine-structural-variation as compared to the reference nucleic acid molecule by comparing the structural information of interest of the GVT-pair on the subject nucleic acid molecule with the corresponding structural information of interest of the segments on the reference nucleic acid molecule.
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Abstract
A method of juxtaposing sequence tags (GVTs) that are unique positional markers along the length of a population of target nucleic acid molecules is provided, the method comprising: fragmenting the target nucleic acid molecule to form target DNA insert; ligating the target DNA insert to a DNA vector or backbone to create a circular molecule; digesting the target DNA insert endonuclease to cleave the target DNA insert at a distance from each end of the target DNA insert yielding two GVTs comprising terminal sequences of the target DNA insert attached to an undigested linear backbone; recircularizing the linear backbone with the attached GVTs to obtain a circular DNA containing a GVT-pair having two juxtaposed GVTs; and recovering the GVT-pair DNA by nucleic acid amplification or digestion with endonuclease having sites flanking the GVT-pair. Cosmid vectors are provided for creating GVT-pairs of ˜45- to 50-kb separation sequencable by next-generation DNA sequencers.
29 Citations
42 Claims
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1. A method for producing juxtaposing sequence tags (GVTs) for determining a fine-structural-variation of a subject nucleic acid molecule, where two constituent members of a sequence tag pair (GVT-pair) located along the length of a population of target nucleic acid molecules are positional markers of a defined separation distance, and the fine-structural-variation is selected from insertion, deletion, duplication, inversion, translocation, or nucleic acid sequence rearrangement;
- wherein the method comprises;
fragmenting the subject nucleic acid molecule to form a target DNA insert; ligating the target DNA insert to a linear DNA backbone at terminal cloning sites of the target DNA insert, leading to the creation of a circular molecule comprising the target DNA insert; digesting the target DNA insert within the circular molecule using at least one endonuclease cleaving the target DNA insert at a distance from each of the insert'"'"'s terminal cloning sites and thereby creating a linear molecule comprising two sequence tags (GVTs) comprising the terminal sequences of the target DNA insert, one of the two GVTs attached to each end of the undigested DNA backbone; recircularizing the linear DNA backbone with the attached GVTs to create a circular DNA molecule thereby creating a GVT-pair comprising two juxtaposed GVTs that are the same relative orientation as the target DNA insert; isolating the created GVT-pair by nucleic acid amplification from primer sites on the DNA backbone or by digestion with endonuclease at sites that are on the DNA backbone and are flanking the created GVT-pair; corresponding the GVT-pair to a reference nucleic acid molecule, wherein each GVT corresponds to a segment of the reference nucleic acid molecule; determining a structural information of interest of the GVT-pair on the subject nucleic acid molecule, wherein the structural information of interest of the GVT-pair on the subject nucleic acid molecule is selected from location of the GVT-pair on the subject nucleic acid molecule, separation distance between the GVT-pair on the subject nucleic acid molecule, or orientation between the GVT-pair on the subject nucleic acid molecule; determining a corresponding structural information of interest of the segments on the reference nucleic acid molecule, wherein the corresponding structural information of interest of the segments on the reference nucleic acid molecule corresponds to the structural information of interest of the GVT-pair on the subject nucleic acid molecule, and the corresponding structural information of interest of the segments on the reference nucleic acid molecule is selected from location of the segments on the reference nucleic acid molecule, separation distance between the segments on the reference nucleic acid molecule, or orientation between the segments on the reference nucleic acid molecule; and determining whether the subject nucleic acid molecule has a fine-structural-variation as compared to the reference nucleic acid molecule by comparing the structural information of interest of the GVT-pair on the subject nucleic acid molecule with the corresponding structural information of interest of the segments on the reference nucleic acid molecule. - 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, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
- wherein the method comprises;
Specification