Who has used Mung Bean Nuclease and How Its effect?! - (Sep/13/2006 )
Who have Used Mung Bean Nuclease? and How is its effect in blunting the Portruding ends of the dsDNA? Many Thanks in advance!
As far as I know, Mung Bean Nuclease is a single-stranded DNA and RNA endonuclease. It removes 3' and 5' extension from DNA or RNA, generating blunt ends. Therefore, it can be used to generate new restriction site and cleavage of hairpin loops.
DNA and RNA, which are in double-stranded form, is resistant to this nuclease.
Hope it helps.
In my hands, Mung Bean S1 Nuclease is easier to use than Nuclease S1. Mung bean is a single-strand-specific nuclease purified from germinated Mung bean sprouts. Degrades DNA and RNA to 5’-phosphoryl mononucleotides. (S. C. Sung and M. Laskowski Sr. 1962 J. Biol. Chem. 237: 506.) Single polypeptide chain MW 39,000. Mung bean nuclease is the enzyme of choice over S1 Nuclease for applications where its low activity on dsDNA is desirable. Ratio of activity on ssDNA versus dsDNA is greater than 1500:1 (measured by hyperchromicity by USB).
Cleaves to leave 3'-hydroxyl on ssRNA or ssDNA template. One unit is defined as the amount of enzyme that produces 1 µg of acid-soluble material per min at 37°C using heat-denatured DNA as substrate. The enzyme is used for the removal of single-stranded extensions (3’ and 5’) to leave blunt ligatable ends, transcription mapping and the cleavage of hairpin loops.
At near neutral pH, mung bean nuclease nicks supercoiled plasmid DNA in unwound regions. Cleavage is not strand-specific and generally occurs only once per supercoiled molecule. (D. Kowalski and J. P. Sanford, 1982. Action of mung bean nuclease on supercoiled PM2 DNA. J. Biol. Chem. 257: 7820-7825.)
In practical terms, I have found Mung Bean S1 to be much more predictable in behavior than S1 nuclease. S1 nuclease has a very narrow window between all and nothing, and is exquisitely sensitive to Zn++ contamination in water. Mung bean nuclease end-nibbles duplex DNA much less than S1 nuclease. (1982 BRL Focus 4 (3) 9) Two units of S1 nuclease removed a terminal labeled nucleotide from 56% of a blunt-ended fragment, while 60 units of Mung Bean Nuclease removed only 24% of the label.
CAUTION: Store in glass if diluted to a concentration below 100 units/µl. At lower concentrations, dilute in 0.001% Triton X-100 to inhibit surface absorption. Glycerol may also be used to reduce the enzyme’s tendency to stick to the sides of the tube.