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METHODS FOR NUCLEIC ACID MAPPING AND IDENTIFICATION OF FINE-STRUCTURAL-VARIATIONS IN NUCLEIC ACIDS

  • US 20090325239A1
  • Filed: 07/10/2009
  • Published: 12/31/2009
  • Est. Priority Date: 01/04/2006
  • Status: Active Grant
First Claim
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1. A method for producing juxtaposing sequence tags (GVTs), 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 or are positional markers of two adjacent and cleavable restriction endonuclease sites of one or more restriction endonucleases, the method comprising:

  • fragmenting a large nucleic acid molecule to form 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 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 ends 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.

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