Figure 2.

Scheme of Limes pathway. One round of Limes can be divided into three different parts: (i) preparation of PCR libraries, (ii) subtractive hybridization and ligation and (iii) purification and amplification of enriched tester sequences. The enriched material can be used for additional rounds of Limes. Initial PCR libraries were prepared by digestion of the subtraction partners with TspRI, by subsequent ligation of PCR-competent flanking sequences (N# and A#) to their ends with the Taq DNA ligase and amplification of the ligation products. The primers (S#) for the library amplification were identical to the respective N# primer apart from the lack of a TspRI recognition site at their 3′-end. In preparation for the subtractive hybridization, the tester library was digested with TspRI again and ligated with a new 5′ flanking N-primer. The driver was digested with SalI. After denaturation and hybridization of tester to an excess of driver, the pool of perfectly matched tester/tester homohybrids containing the tester-specific sequences was ligated with 3′-biotinylated (closed circle) oligonucleotides and purified by separation with streptavidin-coupled beads (horseshoe-shaped magnet) from driver/driver as well as tester/driver hybrids and all kinds of partial hybrids. The purified sequences were amplified with tester-specific S-primers.