Methods for nucleic acid mapping and identification of fine-structural-variations in nucleic acids and utilities
First Claim
Patent Images
1. A method for producing juxtaposed genomic variation tags (GVTs) for determining fine-structural-variations, wherein the juxtaposed GVTs comprise two constituent members of a tag pair (GVT-pair) of a target nucleic acid molecule, the method comprising:
- a) providing a target nucleic acid molecule;
b) fragmenting the target nucleic acid molecule to form one or more fragmented target DNA inserts;
c) providing a DNA adaptor, comprising at least one restriction endonuclease recognition sequence, wherein the restriction endonuclease recognition sequence is located at a predetermined sequence position on the DNA adaptor;
d) ligating a DNA adaptor to each end of the fragmented target DNA insert, thereby forming an adaptor-ligated DNA insert, wherein the location of the restriction endonuclease recognition sequence on each DNA adaptor directs cleavage of the fragmented target DNA insert at a predetermined distance from each end of the fragmented target insert;
e) providing a plasmid vector;
f) ligating each end of the adaptor-ligated DNA insert to one end of the plasmid vector, thereby forming a circular plasmid that carries the adaptor-ligated DNA insert;
g) digesting the circular plasmid that carries the adaptor-ligated DNA insert using restriction endonuclease molecules that recognize the restriction endonuclease recognition sequences on the DNA adaptors, thereby generating a linear molecule comprising two genomic variation tags (GVTs), wherein each GVT comprises a terminal sequence of the fragmented target DNA insert, and each GVT is attached to one end of the plasmid vector via a DNA adaptor;
h) recovering said linear molecule comprising two GVTs;
i) recircularizing said linear molecule by intra-molecular ligation, thereby creating a GVT pair comprising two juxtaposed GVTs;
j) corresponding the GVT pair to a reference nucleic acid molecule, wherein each GVT corresponds to a segment of the reference nucleic acid molecule;
k) determining separation distance and orientation between the GVT pair on the target nucleic acid molecule;
l) determining separation distance and orientation between the segments on the reference nucleic acid molecule; and
m) determining whether the target nucleic acid molecule has a fine-structural-variation as compared to the reference nucleic acid molecule by comparing the separation distance and orientation between the GVT pair with the separation distance between the segments on the reference nucleic acid molecule.
0 Assignments
0 Petitions
Accused Products
Abstract
The invention disclosed herein describes methods for the mapping and identification of fine-structural variations in nucleic acids.
-
Citations
41 Claims
-
1. A method for producing juxtaposed genomic variation tags (GVTs) for determining fine-structural-variations, wherein the juxtaposed GVTs comprise two constituent members of a tag pair (GVT-pair) of a target nucleic acid molecule, the method comprising:
-
a) providing a target nucleic acid molecule; b) fragmenting the target nucleic acid molecule to form one or more fragmented target DNA inserts; c) providing a DNA adaptor, comprising at least one restriction endonuclease recognition sequence, wherein the restriction endonuclease recognition sequence is located at a predetermined sequence position on the DNA adaptor; d) ligating a DNA adaptor to each end of the fragmented target DNA insert, thereby forming an adaptor-ligated DNA insert, wherein the location of the restriction endonuclease recognition sequence on each DNA adaptor directs cleavage of the fragmented target DNA insert at a predetermined distance from each end of the fragmented target insert; e) providing a plasmid vector; f) ligating each end of the adaptor-ligated DNA insert to one end of the plasmid vector, thereby forming a circular plasmid that carries the adaptor-ligated DNA insert; g) digesting the circular plasmid that carries the adaptor-ligated DNA insert using restriction endonuclease molecules that recognize the restriction endonuclease recognition sequences on the DNA adaptors, thereby generating a linear molecule comprising two genomic variation tags (GVTs), wherein each GVT comprises a terminal sequence of the fragmented target DNA insert, and each GVT is attached to one end of the plasmid vector via a DNA adaptor; h) recovering said linear molecule comprising two GVTs; i) recircularizing said linear molecule by intra-molecular ligation, thereby creating a GVT pair comprising two juxtaposed GVTs; j) corresponding the GVT pair to a reference nucleic acid molecule, wherein each GVT corresponds to a segment of the reference nucleic acid molecule; k) determining separation distance and orientation between the GVT pair on the target nucleic acid molecule; l) determining separation distance and orientation between the segments on the reference nucleic acid molecule; and m) determining whether the target nucleic acid molecule has a fine-structural-variation as compared to the reference nucleic acid molecule by comparing the separation distance and orientation between the GVT pair with the separation distance between the segments on the reference nucleic acid molecule. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 30, 32)
-
-
2. A method for producing juxtaposed genomic variation tags (GVTs) for determining fine-structural-variations wherein the juxtaposed GVTs comprise two constituent members of a tag pair (GVT-pair) of a target nucleic acid molecule, the method comprising:
-
a) providing a target nucleic acid molecule; b) fragmenting the target nucleic acid molecule to form one or more fragmented target DNA inserts; c) providing a linear plasmid vector, comprising at least a pair of restriction endonuclease recognition sequences, wherein each restriction endonuclease recognition sequence is located at a predetermined sequence position on each end of the plasmid vector; d) ligating each end of the fragmented target DNA insert to each end of the plasmid vector, thereby forming a circular plasmid that carries the fragmented DNA insert, wherein the fragmented target DNA insert is flanked by the pair of restriction endonuclease recognition sequences, and each endonuclease recognition sequence directs cleavage of the fragmented target DNA insert at a distance from each end of the fragmented target insert; e) digesting the circular plasmid that carries the fragmented DNA insert using restriction endonuclease molecules that recognize the restriction endonuclease recognition sequences on the plasmid vector, thereby cleaving the target DNA insert and creating a linear molecule comprising two sequence tags (GVTs), wherein each GVT comprises a terminal sequence of the target DNA insert, and each GVT is attached to one end of the plasmid vector; f) recovering said linear molecule comprising two GVTs; g) recircularizing said linear molecule by intra-molecular ligation, thereby creating a GVT-pair comprising two juxtaposed GVTs h) corresponding the GVT pair to a reference nucleic acid molecule, wherein each GVT corresponds to a segment of the reference nucleic acid molecule; i) determining separation distance and orientation between the GVT pair on the target nucleic acid molecule; j) determining separation distance and orientation between the segments on the reference nucleic acid molecule; and k) determining whether the target nucleic acid molecule has a fine-structural-variation as compared to the reference nucleic acid molecule by comparing the separation distance and orientation between the GVT pair with the separation distance between the segments on the reference nucleic acid molecule. - View Dependent Claims (19, 31, 33)
-
-
3. A method to create a DNA oligomer through controlled and ordered ligation of short DNA monomers possessing palindromic, rotationally equivalent cohesive ends to yield an oligomeric product bound at both ends by an initiation adaptor, comprising the steps of:
-
forming an oligomer of DNA monomers initiated from an initiation adaptor wherein one adaptor terminus has a nonpalindromic cohesive end that cannot self-ligate, but can adhere to a vector, while the other adaptor terminus has a cohesive unphosphorylated end to prevent adaptor dimer formation and is complementary to the cohesive ends of the DNA monomer for ligation to monomer to initiate oligomer formation; and terminating oligomer growth upon ligation of either a free initiation adaptor to the oligomer formed by adding the DNA monomers or by ligation of another oligomer initiated by the initiation adaptor; wherein the oligomer so formed has an average length regulated by a molar ratio of DNA monomer to initiation adaptor measured when commencing formation of the oligomer.
-
-
20. A method for creating DNA sequence tag oligomers, comprising:
-
a) providing an amount of DNA sequence tag monomers, wherein each DNA sequence tag monomer comprises two cohesive ends, each cohesive end being complementary to a cohesive end of another DNA sequence tag monomer; b) providing an amount of DNA initiation adaptors, each comprising a nonpalindromic cohesive end that cannot self-ligate and an unphosphorylated palindromic cohesive end that is complementary to a cohesive end of the DNA sequence tag monomers, wherein the DNA sequence tag monomers are provided in an excess molar amount as compared to the DNA initiation adaptors; and c) contacting the excess molar amount of the DNA sequence tag monomers with the DNA initiation adaptors, thereby forming an initiation adaptor-ligated DNA sequence tag oligomer comprising a series of DNA sequence tag monomers ligated one to another, and wherein each end of the series of ligated DNA sequence tag monomers is attached to the nonpalindromic cohesive end of a DNA initiation adaptor. - View Dependent Claims (21, 22, 23)
-
-
24. A method for producing a multiplex sequencing vector that carries DNA sequence tags of interest, comprising:
-
a) providing a plurality of DNA sequence tags of interest, b) providing a circular plasmid vector comprising a plurality of vector modules, wherein more than one of said vector modules comprise a type IIS endonuclease recognition sequence and a pair of DNA sequencing primer binding sites for priming Sanger dideoxy sequencing reactions, wherein the endonuclease recognition sequence directs cleavage of those more than one vector modules at a predetermined cleavage site on each module, and each side of said cleavage site is flanked by a DNA sequencing primer binding site; c) digesting the circular plasmid vector using restriction endonuclease molecules that recognize the endonuclease recognition sequence on said more than one vector modules, thereby forming linear plasmid vectors; d) ligating an end of a DNA sequence tag of interest to an end of each linear plasmid vector; and e) reforming a circular plasmid vector from said linear plasmid vectors, thereby generating a circular multiplex sequencing vector that carries a plurality of the DNA sequence tags of interest. - View Dependent Claims (25, 26, 27, 28, 29, 34, 35, 36, 37, 38, 39, 40, 41)
-
Specification