Ribonuclease H cleaves phosphodiester links in the mRNA molecule
DNA polymerase replaces RNA strand with a DNA strand
DNA ligase catalyzes formation of phosphodiester links
Double-stranded DNA molecule
Figure 7.1. Synthesis of a double-stranded DNA molecule.
resistance restriction endonuclease site antibiotic origin of replication gene
the basic components of a typical plasmid cloning vector: an antibiotic resistance gene, a restriction endonuclease site (see next section) at which foreign DNA can be inserted, and an origin of replication so the plasmid can copy itself many times inside the bacterial cell.
The plasmid pBluescript is based on a naturally occurring plasmid that has been engineered to include several valuable features. pBluescript has an origin of replication, and the ampicillin resistance gene for selecting bacterial cells that have taken up the plasmid.The multiple cloning site (MCS) allows the scientist to cut the plasmid with the most appropriate restriction endonuclease for the task at hand.
The MCS lies within the lac Z gene, which codes for the enzyme f-galactosidase (page 112). f-Galactosidase converts a substrate known as X-gal to a bright blue product. Cells containing pBluescript without a foreign DNA in the MCS will produce blue colonies when grown on agar plates. However, when the lacZgene is disrupted by insertion of a foreign DNA in the MCS, the cells containing the recombinant plasmids will grow to produce a colony with the normal color of white. This is because the function of the lac Z gene is destroyed and f -galactosidase is not produced. This is the basis of a test, called the blue/white assay, to identify colonies containing recombinant plasmids.
Another important feature of pBluescript is the presence of the bacteriophage T7 and T3 promoter sequences, which flank the MCS. These promoter sequences are used to transcribe mRNA from a cDNA cloned into one of the sites within the MCS. By selecting the promoter sequence, and adding the appropriate RNA polymerase (T3 or T7 RNA polymerase), either the sense or the antisense mRNA can be synthesized. Antisense RNAs are used in a number of techniques, for example, in situ hybridization (page 147) to detect cells producing a specific mRNA.
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