SECTION 4-1 - pBR322

Abstract

Publisher Summary This chapter discusses pBR322, which is is a 4,362-bp double-stranded DNA plasmid cloning vehicle designed to allow simple and rapid preparation of cloned recombinant DNA fragments. It contains two antibiotic resistance genes, an origin of replication, and a variety of useful restriction sites for cloning or subcloning restriction fragments. pBR327 is identical to pBR322, except that it is missing all nucleotides between 1,427 and 2,516. These sequences are deleted to reduce the size of the cloning vector and to eliminate sequences, which interferes with the expression of cloned DNA in eukaryotic cells. pBR322 and pBR322-derivative plasmids are very common plasmid vectors. These plasmid cloning vectors confer antibiotic resistance when E. Coli—deficient in the resistance genes—are successfully transformed. The two antibiotic resistance genes possess unique cloning sites for the insertion of DNA fragments. Opening the plasmid with an RE and inserting a compatible DNA segment with DNA ligase inactivates this resistance gene. Selection is achieved by growing and plating the transformed cells in the presence of the other antibiotic.