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

About Author

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Nidhi Abhay Kulkarni

Nidhi Abhay Kulkarni completed her BSc. Microbiology from Savitribai Phule Pune University. She has published two articles in the Scientific Journal. She is interested in research related to medical microbiology, molecular biology, and genetics. She also has good Laboratory and Bioinformatics skills.

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