Mung bean nuclease

Requirements

Mung bean nuclease requires Zn2+. The addition of EDTA or SDS causes irreversible inactivation. Mung bean nuclease is not active at pH below 4.6, nor at low salt concentration.

Description

Nuclease MB is a specific DNA and RNA exo-endonuclease which will degrade single-stranded extensions from the ends of DNA and RNA molecules, leaving blunt, ligatable ends. Its higher single-strand specificity makes it the enzyme of choice for most applications requiring a single-strand-specific nuclease.

Unlike S1 Nuclease, Mung Bean Nuclease will not cleave the intact strand of nicked duplex DNA.

Its ability to recognise double-stranded nucleic acids depends on the base sequence.

It tends to cleave at ApN and at T(U) pN. It completely degrades ApA, but does not degrade G and C. Unlike S1 Nuclease, it does not cleave the strand opposite to that which has been nicked.

Mung Bean Nuclease catalyzes the specific degradation of single-stranded DNA or RNA, and produces mono- and oligonucleotides carrying a 5′-P terminus.

More than 1000- fold amount of enzyme can degrade oligomer into all mononucleotides.

An excess of the enzyme is required to degrade double-stranded DNA or RNA and DNA-RNA hybrids, and in this case, AT-rich regions are selectively degraded.

This enzyme work well at A↓pN, T ↓pN sites, and especially A↓pN sites are 100% degraded.

However, it is difficult to degrade C↓pC, C↓pG site.

Mung bean exonuclease is a nuclease derived from mung beans that removes nucleotides in a step-wise manner from single stranded DNA molecules and is used to remove such ssDNA from a mixture also containing double stranded DNA (dsDNA).

Unit Definition:

One unit of Mung Bean Nuclease converts 1 µg of heat-denatured calf thymus DNA into an acid-soluble form in 1 minute at 37 °C under standard assay conditions.

Applications in biotechnology and biochemical research

• Removal of hairpin loops during cDNA synthesis.
• High-resolution mapping of the termini and exon structures of RNA transcripts (commonly termed Berk-Sharp or S1 Mapping) using either internal-labelled or end-labelled probes.
• Restriction-site modification or removal by digestion of single-stranded protruding ends.
• Cleavage of single-basepair mismatches, as a replacement for CEL 1 Nuclease in TILLING.
• Unidirectional deletion of large DNA (in combination with Exonuclease III) to generate ordered deletions for sequencing.
• Removal of 3´ and 5´ extensions from DNA or RNA termini.
• Transcriptional mapping.
• Cleavage of hairpin loops.
• Excision of gene coding sequences from genomic DNA.

References

References

1. "BRENDA: 3.1.30.1".
2. (1996). "Enzymology primer for recombinant DNA technology". Academic Press.