Alkaline phosphatase removes 5' phosphate groups from DNA and RNA. It will also remove phosphates from nucleotides and proteins. These enzymes are most active at alkaline pH - hence the name.
There are several sources of alkaline phosphatase that differ in how easily they can be inactivated:
- Bacterial alkaline phosphatase (BAP) is the most active of the enzymes, but also the most difficult to destroy at the end of the dephosphorylation reaction.
- Calf intestinal alkaline phosphatase (CIP) is purified from bovine intestine. This is phosphatase most widely used in molecular biology labs because, although less active than BAP, it can be effectively destroyed by protease digestion or heat (75C for 10 minutes in the presence of 5 mM EDTA).
- Shrimp alkaline phosphatase is derived from a cold-water shrimp and is promoted for being readily destroyed by heat (65C for 15 minutes).
There are two primary uses for alkaline phosphatase in DNA manipulations:
- Removing 5' phosphates from plasmid and bacteriophage vectors that have been cut with a restriction enzyme. In subsequent ligation reactions, this treatment prevents self-ligation of the vector and thereby greatly facilitates ligation of other DNA fragments into the vector (e.g. subcloning).
- Removing 5' phosphates from fragments of DNA prior to labeling with radioactive phosphate. Polynucleotide kinase is much more effective in phosphorylating DNA if the 5' phosphate has previously been removed.
It is usually recommended that dephosphorylation of DNAs with blunt or 5'-recessed ends be conducted using a higher concentration alkaline phosphatase or at higher temperatures than for DNAs with 5' overhangs.
|
