Patent ID: 6929917
Filing Date: 2005-08-16
Classification: C12N

Abstract:
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.