FIG. 6.

Repair of a DSB by allelic recombination does not result in detectable levels of crossing over between alleles. (A) Two potential crossover intervals are indicated: co1, positioned between the DSB and the P6 marker, and co2, positioned between the P6 and St markers. (B) Recombinants were analyzed for a crossover in interval co1, which would establish a new linkage of the P6+ polymorphism of the S allele to the PacI restriction site polymorphism of the P allele, as shown in the diagram. This novel linkage was assayed for by PCR amplification using the primers rb1 and p6+, the latter of which specifically amplifies alleles containing the P6+ polymorphism, followed by cleavage of the product by EagI (Ea) or PacI (P) and resolution of the products on an agarose gel along with uncut product (U). The products from the parental cell line (S/P) and a representative recombinant clone (R) are shown. None of the recombinants showed this novel linkage. (C) Recombinants with LOH at the p6 marker, but not the St marker, were analyzed for the product of a crossover in interval co2, which would establish a novel linkage of the StuI restriction site polymorphism of the S allele to the PacI restriction site polymorphism of the P allele, as shown in the diagram. Assays to detect the presence of this novel linkage were performed by PCR amplification of the neo gene from the 4- to 5-kb size-enriched StuI fragments from genomic DNA. The neo genes were amplified using the primers ep1 and ep2, and the products were subsequently digested by EagI (Ea) or PacI (P) and resolved on an agarose gel along with uncut product (U). The products from the size-enriched genomic DNA (StuI; 4- to 5-kb size selected) derived from the parental cell line (S/P) and a clone (R) which is representative of the recombinants tested for this novel linkage are shown. Also shown are the products from the total genomic DNA of the parental cell line.