**3.3.2 Genotyping by PCR or southern blotting**

Screening selected colonies by PCR for a gene targeting event typically involves using one PCR primer specific to the host genome sequence and the other primer specific to the selectable marker between the arms of homology in the targeting vector. This approach provides a simple, rapid and highly sensitive method to identify the gene targeting event in the transfected cells (Gómez-Rodríguez et al., 2008). Furthermore, PCR can be performed using the lysate from the small amount of cells, and also can be facilitated by pooled analysis of multiple cell lines. These types of PCR analysis sometimes require optimization as the amplicon is typically several thousand bp depending on the length of the targeting arms. Following the initial screening via PCR, the targeted colonies can be further expanded for analysis by southern blotting. A strategy for identifying targeted cells by southern blotting should be incorporated in the vector design. Two DNA probes on each side of the wild type gene, but outside the targeting vector, are designed to detect a change in fragment size resulting from either introduction or elimination. It is necessary to verify that the probes for southern blotting work well on wild-type genomic DNA from the somatic cells before the targeting experiment. Also, it is important to perform southern blotting analysis using both probes to confirm the double-crossover event since homologous recombination can occur at only one end of the targeting vector (Hasty et al., 1991b; Moens et al., 1992). Of the methods of screening for gene targeting in somatic cells, southern blotting is considered to be the golden approach to identify correctly targeted colonies despite being time-consuming and relatively expensive compared to PCR approaches.
