A simplified, orientation-specific cDNA cloning strategy.

D) DIGESTION WITH Hind III AND EcoRI PROTOCOL, a) cDNA synthesis by Reverse Transcriptase is initiated with oligo(dT) according to Gubler and Hofmann (1) . b) The double stranded cDNA is methylated, and (c) a synthetic CCTTGAATTCAAGCNH oligonucleotlde pGCTTGAATTCAAGC CCAACTTAACTTCCNN is added. This generates a Hindlll recognition sequence (AAGCTT) when ligated to the oligo (dA) track at the 3 terminal end of the cDNA molecule and an EcoRI site at the 5' terminus. In our protocol, the cDNA should be methylated with M.EcoRI and M.Hindlll (2). Since M.Hindlll was not commercially available, M.AluI (New England Biolabs) was tested and shown to provide complete protection of lambda DNA against Hindlll digestion, d) The ligation mixture is digested with Hindlll and subsequently with EcoRI, in order to prevent inefficient cleavage of the terminal Hindlll site, e) After removal of excess linker molecules (3) the cDNA is inserted in a specialized vector. AATTCAACCHNCTTCCNN -