PstI

PstI is a type II restriction endonuclease isolated from the bacterium Providencia stuartii. It recognizes the six-base pair palindromic sequence CTGCAG in double-stranded DNA and cleaves near this site to generate cohesive, or sticky, ends. PstI is widely used in molecular biology as a tool for DNA cloning and analysis.

The enzyme creates staggered cuts at its recognition site, producing ends that can anneal with complementary sequences generated by other restriction enzymes. This property makes PstI useful for directional cloning, where the orientation of an insert can be controlled relative to a vector. In practice, PstI digestion is often combined with other enzymes to generate compatible ends for ligation or to map and analyze DNA fragments.

Reaction conditions and limitations are important for effective use. PstI operates optimally at around 37°C in appropriate buffer systems, with performance influenced by factors such as ionic strength and DNA methylation. Methylation of the recognition sequence can inhibit digestion, so unmethylated DNA is typically required for efficient cleavage. Commercial suppliers provide standardized buffers and guidelines to maximize reliability.

History and significance: PstI has been a staple in molecular cloning since the early development of restriction enzyme technology. It remains a standard choice for constructing recombinant DNA molecules and for various genome analysis tasks, contributing to advances in genetics, biotechnology, and education.

See also: restriction enzymes, cloning vectors, DNA ligation.