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Method for cloning of a rare, specifically mutated cell

  • US 6,929,917 B2
  • Filed: 11/18/2002
  • Issued: 08/16/2005
  • Est. Priority Date: 11/18/2002
  • Status: Expired due to Fees
First Claim
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1. A method of detecting and isolating a cell in a culture, which cell is mutated by a sequence-specific process, the method 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 allele-specific polymerase chain reaction (AS-PCR) primer that encodes the 3′

    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, wherein the sequence-specific process is short fragment homologous replacement (SFHR), the SFHR comprising the use of an exogenous nucleic acid having a sense strand, an antisense strand, or both.

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