Figure 1

Primer Design for 3C Experiments. (a): The important steps of Chromosome Conformation Capture (3C): (1) Interacting DNA segments (red and black) are crosslinked. (2) Crosslinked DNA segments are cut with a restriction enzyme (EcoRI in this example, restriction sites shown as green lines). (3) Segments are religated at low DNA concentration strongly favoring intramolecular ligations between crosslinked segments compared to other, free floating segments. (4) Crosslinks are removed. (5) Hybrid templates are purified and quantified by quantitative PCR. After digestion (step 2), this figure suggests the possibility of four unique ligation products of the four DNA ends - each resulting in a ligation product between the red and the black fragment. Since only upstream primers are designed and used for the 3C method itself, out of these four products, only one is amplified. Positions of possible upstream primers are denoted by black arrows. Downstream primers, as designed by our program should be used to ckeck proper digestion of each site to be studied! ( b): Graphical representation of some primer search parameters. Minimum and maximum amplicon lengths are shown depending on minimum and maximum primer lengths. All parameters are specified by the user. ( c): From the list of candidate primers as obtained in (b), for each scan region, primer tuples are created, checked and scored. This figure shows the optimal primer pair for the current restriction site. Primers and primer tuples are scored by the scoring functions depicted in Figure 3. (d): In order to retrieve the optimal set of primer tuples, one for each sequence region to be scanned, the lists of acceptable primer tuples (shown as columns) are screened. A candidate primer tuple set corresponds to a path through the matrix of candidate tuples from left to right. It should be noted that this tuple set contains exactly one primer tuple for each scan region to be screened. In each step, the primer tuple which is most homogeneous to the current set is added. At the end, the tuple set is scored and the optimal scoring tuple set is returned.