Cohesive termini

Bacteriophage X is a genetically complex and extensively studied virus of E. coli. Because it has been the object of molecular genetic research, it was investigated and developed as a vector. The DNA of phage X, in the form in which it is isolated from the phage particle, is a linear duplex molecule of about 45.5 kb pairs. The entire DNA sequence has been determined (SI). At each end are short, single-stranded 5 projections of 12 nucleotides that are complementary in sequence and by which the DNA adopts a circular structure when it is injected into the host cell i.e., X DNA naturally has cohesive termini that associate to form the cos site. 

Use of Primer-Adapter and the Generation of Cohesive Termini of cDNA... 

When the ability of nucleases SI and MB to trim the single-stranded portion of the cohesive termini of X DNA was compared (25), nuclease SI (at pH 4.5) made a complete cleavage of the 12-nt ends at 10°C as well as at 30°C without any digestion of the dsDNA. Nuclease MB (at pH 5.0) cleaved off the single-stranded ends completely at 30°C but, at 5°C, 4 nt remained undigested even at high enzyme concentrations. Under both reaction conditions, nuclease MB introduced some nicks into dsDNA. 

Except for some special applications (see below), the conventional linker method of cloning is now largely superseded by a simpler and more efficient adapter method. In this improved method, two synthetic oligonucleotides are annealed to form a restriction site (e.g., EcoRI) in such a way that it has the necessary cohesive termini on one end, while the other end is blunt-ended and 5 -phosphorylated to allow ligation to the DNA (20,21). 

V. Blunt-end formation of duplex DNA. Duplex DNAs with cohesive termini can be blunted by using Pol Ik in one of the following ways. 

When the DNA carries 3 -recessed ends, they can be filled in by the polymerase reaction in the presence of appropriate dNTPs. In the presence of limited, selectively complementary dNTPs, partial filling in can be obtained, allowing versatile use of the partially cohesive termini. 

Fig. 4. A model of the nucleotide sequence arrangement contained within AAV DNA, Two nucleotide sequence permutations are illustrated. Plus and minus strands may anneal to form duplex linear monomers with (3 and 4) or without cohesive 3 or 5 termini (1 and 2). Duplex linear monomers with cohesive termini can then form duplex circular monomers or duplex linear oligomers. In the figure the terminal repetitions are depicted as symmetrical nucleotide sequences. In the inset two alternative types of terminal repetitions are illustrated the first has the inverted repetition subterminal to the natural repetition, the second illustrates the possibility that a strand may have either an inverted or a natural terminal repetition...

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