pUC19 Vector- Definition, Structure, Sites, Applications

pUC plasmids were first developed by Joachim Messing and his co-workers. It is a commonly used cloning vector in the bacteria E. coli

pUC19 is 2686 bp in length. The molecular weight of the pUC19 vector is 1.75×106 Da. It is a small plasmid with a high copy number. It contains the lacz gene and has multiple cloning sites. 

Hence, it is widely used as a cloning vector. pUC19 plasmid is similar to pBR322 plasmid in structure.

pUC19 full form

p = plasmid

UC = University of California

19 = numerical designation

pUC19 Vector
pUC19 Vector

Structure of pUC19

  • It is a 2686 bp long plasmid.
  • Origin of replication – The origin of replication of the pUC19 plasmid is derived from pMB1. 
  • Multiple Cloning Sites –  There is a short sequence of 2.8 kb which contains sites for various restriction enzymes. This increases the number of potential restriction sites available, enabling the production of the desired fragment for cloning. 
  • Selectable markers – The pUC19 plasmid contains an Ampicillin resistance gene which can be used to screen the recombinants. The plasmid also contains the E. coli gene lacZ, which encodes for β-galactosidase (β-galactosidase hydrolyses lactose).
  • Restriction sites – The pUC19 vector carries a 54 bp long multiple cloning site poly-linker containing 13 different hexanucleotide-specific restriction endonucleases sites. 

Some of the restriction sites are EcoR1, HindIII, BamH1, and many more.

Figure: pUC19 map. Image Source: NEB.

Screening of pUC19 vector

The Blue-White screening method is used for pUC19 vectors. The process of screening is as follows:

  • This method of screening is based on the fact that the blue pigment is formed when β-galactosidase catalyzes the hydrolysis of a synthetic substance known as X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside) in the medium. 
  • When X-gal is hydrolyzed, it forms galactose and 5-bromo-4-chloro-3-hydroxy indole. 
  • The later product undergoes dimerization (spontaneous) and oxidation. 
  • As a result of dimerization and oxidation, a blue pigment is formed. 
  • The cells which contain the β-galactosidase activity form blue colonies, whereas the cells which do not show β-galactosidase activity form white colonies on the agar medium containing X-gal. 
  • The recombinant cells, which contain newly inserted DNA fragments, lack the β-galactosidase activity and hence appear white on the agar plates.
  • This method of screening the recombinant cells is the easiest and fastest method. 

Advantages

  • This is a small cloning vector and has large industrial applications. 
  • It has one step selection process for the recombinants, hence is used on a large scale. 
  • It has a high copy number. 
  • The presence of many restriction sites makes it suitable for cloning. 

References

  1. https://www.snapgene.com/resources/plasmid-files/?set=image_consortium_plasmids&plasmid=pUC19 
  2. PRINCIPLES OF GENE MANUPULATION AND GENOMICS BY PRIMROSE AND TWYMAN 
  3. https://enzyquest.com/product/puc19-dna-plasmid/ 
  4. Julin, D.A. (2018). Blue/White Selection. In: Wells, R.D., Bond, J.S., Klinman, J., Masters, B.S.S. (eds) Molecular Life Sciences. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-1531-2_94

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