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Methods for nucleic acid mapping and identification of fine-structural-variations in nucleic acids and utilities

  • US 7,932,029 B1
  • Filed: 01/03/2007
  • Issued: 04/26/2011
  • Est. Priority Date: 01/04/2006
  • Status: Active Grant
First Claim
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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.

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