Figure 4

Detection of heterozygous condition. A: a. BTH102 were transformed with pREAL/WT:pREAL/NS in a 1:1 proportion to test the detection system for heterozygous PCR product. Gray arrows show white colonies. b: BTH102 were transformed with pREAL/WT as homozygous control. B: localization of the primers (arrows in gray) used to amplify DNA fragments from red and white colonies to detect heteroduplex formation. C: DNA was amplified directly from colonies. Red and white colonies were touched with a pipette tip, added to the same PCR mix for amplification, afterwards heat denatured, and allowed to re-hybridize at low temperature. The obtained products were run on a non-denaturing 8% PAGE. In lane 2, the appearance of two slowly migrating bands indicates the presence of heteroduplexes (black arrows) in the mixed sample (WT/NS product). Lanes 1 and 3 are non-combined red and white colonies amplified in different PCR mix, used as controls. In these lanes, the heteroduplex bands are absent.