By Leonard Davis (Auth.)
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The cleavage site specificities for many REs have b e e n defined. F o r exam ple, t h e RE EcoRl requires that six base pairs occur in t h e following specific order: 5 ' . . GAATTC. . 3 ' 3 ' . . CTTAAG. . 5 ' EcoRl recognizes this sequence and cleaves it in a unique fashion, resulting in t w o termini with protruding 5' ends: . G . CTTAA and 51 AATTC. . G. . 5 I Preparation of DNA from Eukaryotic Cells These e n d s are complementary ("sticky") and can b e enzymatically reat tached t o any other IfcoRI-generated termini using T4 DNA ligase.
It is m o s t useful a s a cloning vector for generating genomic libraries after partial Mbol digestion. This cloning vector can a c c o m o d a t e u p to approximately 23-kb inserts, making it suitable for genomic library construction. The design of t h e polylinker s e q u e n c e s simplifies preparation of t h e vector for cloning partial Mbol fragments 15-20 kb in size. In addition, t h e polylinker s e q u e n c e s allow easy removal of cloned DNA at the cloning site. This double-stranded, 44-kb DNA bacteriophage g r o w s well on appropriate E.
0 — BgtlL Bom H I Sma I BomHl BglS HindTB. 7 Xhol BomHl Hindis EcoRl Bg/U Sma I BomHl Hindis. 4 3 . 4 Diagram of XgtlO, showing major RE sites. 7-kb linear double-stranded X bacteriophage cloning vector de signed exclusively for cloning small EcoRl fragments (less t h a n 6 kb), in particu lar cDNA fragments with EcoRl linkers. The unique EcoRl site u s e d for inserting foreign DNA is located n e a r t h e COOH t e r m i n u s of t h e lac Ζ gene. Foreign DNA s e q u e n c e s in this cloning vehicle can b e e x p r e s s e d as β-galactosidase fusion proteins.