089

Klonowanie DNA przy pomocy wektora wywodzącego sie od bakteriofaga PI

Plasmid

1. BamH I + Scal

i ',¹¹-

2, Ligate to

DNA

Insert DNA

P1 recombinant

75-100 kb

Figurę 4.18: The phage P1 vector system allows DNA fragments of up to 100 kb to be cloned.

The P1 plasmid vector Ad10 incorporates various elements of the P1 genome, notably pac, the P1 packaging site, and two /oxP sites, which are the sites naturally recognized by the phage recombinase, the product of the cre gene. The vector is digested so as to generate two arms, a short arm and a long arm to which 85-100 kb size-selected foreign DNA fragments are ligated. Packaging of the recombinant DNA occurs in vitro using P1 packaging extracts: pacase cleaves the recombinant DNA at the pac site and then works with other components to insert the DNA (maximum of 115 kb) into phage heads. Taił proteins are attached and the recombinant phage is allowed to adsorb to a cre⁺ strain of £ coli. The host celi cre product acts on the /oxP sites so as to produce a circular plasmid which is maintained at Iow copy number (by the plasmid replicon) but can be amplified by inducing the P1 lytic replicon.