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Method for cloning of a rare, specifically mutated cell

  • US 20040096832A1
  • Filed: 11/18/2002
  • Published: 05/20/2004
  • Est. Priority Date: 11/18/2002
  • Status: Active Grant
First Claim
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1. In a method of making a specific directed mutation in the genome of a cell by a sequence-specific process, of the type wherein a rare cell in culture is mutated by the sequence-specific process and the rare mutated cell is cloned from the culture, the improvement comprising:

  • a) treating the culture with a sequence-specific process that introduces a 3′ and

    a 5′

    mutation in a genomic target, wherein the 3′ and

    5′

    mutations are separated by not more than 100 nucleotides;

    b) forming replicate subcultures of the treated culture;

    c) making a first polymerase chain reaction (PCR) product that contains the site of the 3′ and

    5′

    mutations using a sample of genomic DNA from a replicate of at least two subcultures of the treated culture;

    d) making a second PCR product that contains the site of the 5′

    mutation using the first PCR product, an AS-PCR primer that encodes to the 3′

    nucleotide mutation and a forward primer that is not homologous to the site of the 5′

    mutation;

    e) identifying a positive subculture by the presence of the 5′

    mutation in the second PCR product using template from the subculture;

    f) subdividing a replicate of the positive subculture;

    g) identifying a positive subdivision of the positive subculture by the presence of the 5′

    mutation in cells of the subdivision; and

    h) verifying that the positive subdivision contains a doubly substituted cell having both the 3′ and

    5′

    mutations and cloning the doubly substituted cell.

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