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Pac vector ppt

p1 derived artificial chromosome (pac) vector

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Pac vector ppt

  1. 1. Pronabjyoti Mahatta. M.Sc. 4th semester Microbiology Roll No. 10 P1 derived artificial chromosome (PAC) vector
  2. 2. INTRODUCTION  A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell into which the DNA fragment to be cloned (DNA insert) is integrated for cloning.  The vectors used for propagation of DNA inserts in a suitable host are called cloning vectors.  There are many vectors which are developed time to time for different purposes.
  3. 3.  Plasmid vectors  pSC101, pBR322, pUC18, pUC19, Pgem3z  Bacteriophage vectors  Lambda phage vectors, phage M13 vectors  Cosmid vectors  pJB8  Phagemid vectors  pBluescriptSK  Phasmid vectors  Lamba ZAP  Artificial chromosome vectors  PAC, BAC, YAC.
  4. 4. PAC VECTOR OR PHAGE P1 VECTOR  Phage P1 is a temperate bacteriophage which has been extensively used for genetic analysis of Escherichia coli because it can mediate generalized transduction.  Sternberg and co-workers have developed a P1 vector system which has a capacity for DNA fragments as large as 85 - 100 kb. (sternberg 1990, Pierce et al.1992)
  5. 5.  The capacity is about twice that of cosmid clones but less than that of yeast artificial chromosome (YAC) clones.  P1 vector contains a packaging site (pac) which is necessary for in vitro packaging of recombinant molecules into phage particles.  The vectors contain two loxP sites. These are the sites recognized by the phage recombinase, the product of the phage cre gene, and which lead to circularization of the packaged DNA after it has been injected into an E. coli host expressing the recombinase .
  6. 6.  Clones are maintained in E. coli as low-copy-number plasmids by selection for a vector kanamycin- resistance marker.  A high copy number can be induced by exploitation of the P1 lytic replicon.  This P1 system has been used to construct genomic libraries of mouse, human, fission yeast, and Drosophila.
  7. 7. DESCRIPTION OF THE DIAGRAM :  The phage P1 vector system. The P1 vector Ad10 (Sternberg 1990) is digested to generate short and long vector arms. These are dephosphorylated to prevent self-ligation. Size-selected insert DNA (85– 100 kb) is ligated with vector arms, ready for a two- stage processing by packaging extracts.  First, the recombinant DNA is cleaved at the pac site by pacase in the packaging extract.
  8. 8.  Then the pacase works in concert with head/tail extract to insert DNA into phage heads, pac site first, cleaving off a headful of DNA at 115 kb. Heads and tails then unite. The resulting phage particle can inject recombinant DNA into host E. coli. The host is cre+. The cre recombinase acts on loxP sites to produce a circular plasmid.  The plasmid is maintained at low copy number, but can be amplified by inducing the P1 lytic operon.