pUC vectors used as cloning vectors and they belong to pUC series (named after the place of their initial preparation i.e. University of California). These plasmids are 2700 bp long and contains (Figure 16):
- Ampicillin resistance gene
- Origin of replication derived from pBR322,
- lacZ gene derived from E. coli. Within the lac region is also found a polylinker sequence having unique restriction sites (identical to those found in phage M13), and
- Multiple cloning sites (MCS).
These plasmids when transformed into an appropriate E.coli strain having lac (e.g. JM103, JMI09), and grown in the presence of IPTG (isopropyl thiogalactoside, which behaves like lactose, and induces the synthesis of f3 galactosidase enzyme) and X-gal (substrate for the enzyme), will give rise to white or clear colonies.
On the other hand, pUC having no inserts and transformed into bacteria will have an active lacZ gene and therefore will produce blue colonies, thus allowing identification of the colonies having pUC vector with cloned DNA segments (figure 17).
As discussed above, in pBR322 and pBR327, the DNA is inserted at a site located in one of the two genes for the resistance against antibiotics, so that it inactivates one of the two resistance genes.
The insert bearing plasmid can be selected by their ability to grow in a medium containing only one of the two antibiotics and by their failure to grow in a medium containing both the antibiotics.
Plasmids carrying no insert on the other hand, grow in media containing one or both the antibiotics. So, the presence of lacZ gene in pUC and resistance genes against ampicillin and tetracycline in pBR322 and pBR327 allow selection of E.coli colonies transformed with plasmids carrying the desired foreign cloned DNA segment.
pUC19 is a commonly used high copy number cloning vector. The vector encodes the N-terminal fragment of β-galactosidase (lacZa), which allows for blue/ white colony screening (i.e., a-complementation), as well as a pUC origin of replication and an ampicillin resistance gene that allow propagation and selection in E.coli.
Important feature of pUC plasmids is blue/white colony screen to detect recombinant plasmids. This screen is based upon inactivation of the lacZa peptide of beta-galactosidase, which is expressed by the vector.
The cloning vectors belonging to pUC family are available in pairs with reversed orders of restriction sites relative to lacZ promoter. pUC8 and PUC9 make one such pair. Other similar pairs include pUC12 and pUCl3 or pUC18 and pUC19.
Drawbacks of pUC Vectors
The foundation of genomic sequence analysis is large-scale cloning and sequencing from shotgun plasmid libraries obtained by assembling sequences from vast majority of clones.
- Gaps in shotgun libraries and unclonable DNA fragments are quite common. DNA is characterized by high AT content, strong secondary structure, open reading frames, or cis-acting functions (e.g., transcriptional promoters or replication origins).
- In some cases, most notably AT-rich DNA, the reasons for difficulty in cloning are not well defined.
- One or more features of pUC plasmids have been shown to be incompatible with cloning or stable maintenance of the inserts, which may lead to severe difficulties in creating plasmid libraries, especially from DNA rich in AT bases (>70%).