Primer design. Depicted is an actual case of the Chlamydomonas RDP3 target gene (ID, 183511) interrupted by the marker gene. The marker gene consisted of a PCR-amplified fragment containing the AphVIII gene under the control of the PSAD promoter, and with an incomplete 3'UTR. Since the DNA marker can be integrated into the Chlamydomonas genome in two different orientations, two forward (F1 and F2) and two reverse target gene primers (R1 and R2) were used to identify the insertion site. Typically, forward and reverse primers are separated by ~1.0 kb along the sequence of the target gene. Each target gene primer is used in combination with individual marker gene primers (RB1 and RB2) in independent PCR amplifications. Amplification would only be possible in those transformants in which the marker gene is inserted close to or within the target gene. In the example depicted, the marker gene was inserted within the 5'UTR of the target gene and amplification was observed with the RB2-R1 primer pair. Exons, introns and UTR regions are represented by black, white and grey squares respectively.