Cloning of region-specific genetic markers of planarian Dugesia tigrina bymeans of a new method: Ordered differential display

A new method for finding; differentially expressed genes, termed ordered di fferential display of mRNAs (ODD), was used in the search for region-specif ic molecular markers of freshwater planarian Dugesia tigrina. In this metho d, the effect of selective suppression of a polymerase chain reaction (PCR) is used for the differential amplification of a pool of 3'-terminal cDNA f ragments generated by digestion of cDNAs with a restriction endonuclease. I n the resulting amplified cDNAs, every mRNA is represented by a cDNA fragme nt whose. length is determined by the position of the restriction site near est to the 3'-terminus. Subsequent PCR with primers 3'-extended by two rand om nucleotides;:allowed the amplification of 1/192 part of all cDNA molecul es present in the sample. The comparison of the generated pools of cDNA mol ecules separated by PAGE leads to the identification of differentially expr essed sequences. The systematic study of the total mRNA pool is achieved by the successive use of all possible combinations of extended primers. Some sequences preferentially expressed along the anterior-posterior axis of pla narian were identified using ODD.