Insertion Vectors

Insertion Vectors

Vectors that have a single target site for insertion of foreign DNA are known as insertion vectors. 20% DNA that is not required for lytic growth is removed and therefore insertion of foreign DNA resumes the size back to something like its full length and can be packaged in vitro. Maximum size of DNA that can be accommodated varies from 9- 11 kb. For DNA of larger sizes, high capacity vectors are designed like:-

Vector                size.                     OrI                            Host
Cosmid.            30-45kb.           Col E1                           E.coli
BAC.                 120-300kb.          Replicon forgein      E.coli  
YAC                   250-400kb           ARS                            Yeast

What is Bacteriophage Artificial Chromosomes (BACs) ?

What is Bacteriophage Artificial Chromosomes (BACs) ?

These plasmids are circular DNA molecules carrying conventional antibiotic resistance marker, origin of replication derived from the F factor of E.coli, an ATP driven helicase (repE) to facilitate DNA replication and three loci (parA, parB and parC) for proper partitioning of the plasmid to daughter cells. BAC vectors have no packing constraints and there is no fixed limit to the size of genomic DNA that they accept. Usually the size of DNA is approximately 120-kilo base pairs.  

Yeast Artificial Chromosome (YACs)

Yeast Artificial Chromosome (YACs)

These are linear DNA molecules similar to yeast chromosome. Recombinant YACs are made by ligating large fragments of genomic DNA and then the resultant plasmid is introduced into yeast by transformation. The vector carries selection marker, DNA sequences called as telomere, so that the product can be stabilized inside the yeast cell, an origin of replication called autonomous replication origin, ARS. Large size of DNA can be inserted into YAC vectors, usually between 250kilobases to 400kilobasepairs. Large size of mammalian genomic libraries is also made with approximately 1 megabasepairs of foreign inserts. Insertion of foreign DNA into the cloning site inactivates a mutant expressed in vector DNA and formation of red rather than white colonies by yeast strain is observed. Thus transformants are identified as red colonies, which grow in yeast that is mutant for TRP1 and URA3, which ensure that the cell has received an artificial chromosome and with both the telomeres since it is complimented for both the mutations. And the colony also contains foreign DNA because it is red in color.