Mismatch Endonuclease I is a Mg2+ dependent DNA endonuclease that specifically cleaves mismatched base pairs (T:T, G:G and T:G mismatches). Mismatch Endonuclease I cleaves the 3rd phosphodiester bond on the 5´ side of the mismatched base in both strands, leaving a 5-base pair overhang. While Mismatch Endonuclease I prefers to cleave T:T, G:G and T:G DNA mismatches, it will also readily cleave T:I, G:I and G:U DNA mismatches. Additionally, Mismatch Endonuclease I has been shown to nick the thymine containing strand of T:C DNA mismatches, and cleave T:G and T:U (DNA:RNA) mismatches albeit to a lesser extent than DNA:DNA mismatches.Figure 1. Mismatch Endonuclease cleaves T:T, G:T and G:G mismatches in dsDNA.(A) Four plasmid constructs (p1-p4) containing a specific mutation at the same locus and differentially phosphorylated primers (either forward or reverse) were used to generate eight double-stranded PCR generated fragments ~672 bp in length (ds1-ds8). Following cleanup (Monarch PCR & DNA Cleanup Kit (NEB #T1030)), the phosphorylated strand of the double-stranded fragments (2.2 µg) was specifically degraded using 5 units of Lambda Exonuclease, incubated at 37°C for 60 minutes, to generate single-stranded oligos of either the top or bottom strand containing either A, T, C or G at the same locus. The single-stranded oligos (ss1-ss8) were then purified using the Oligonucleotide Cleanup Protocol for the Monarch PCR & DNA Cleanup Kit (NEB #T1030). (B) Purified single-stranded oligos containing either an A, T, C or G can then be mixed and matched to form either perfectly Watson-Crick base-paired DNA (green check marked boxes) or double-stranded DNA oligos containing a single-base mismatch (yellow X boxes). Mismatched dsDNA oligos were generated by mixing the appropriate top and bottom strands (for the mismatch to be created) and re-annealing in 1X NEBuffer 2.1, heated to 95°C, followed by cooling to room temperature, to generate the eight potential DNA mismatches (A:A, A:C, A:G, C:C, C:T, G:G, G:T, T:T). (C) The ability of Mismatch Endonuclease I to cut specific mismatches in dsDNA was queried by incubating 80 units of Mismatch Endonuclease I with 200 ng of mismatch containing dsDNA, incubated at 37°C for 30 minutes. Products were then run on a 1.2% agarose gel and visualized with ethidium bromide staining.Figure 2. Mismatch Endonuclease cleaves T:T, G:T and G:G mismatches in dsDNA.The ability of Mismatch Endonuclease I to cut specific mismatches in dsDNA was queried by incubating 80 units of Mismatch Endonuclease I (+M0678), incubated in NEBuffer r2.1 at 37°C for 30 minutes, with double-stranded DNA oligonucleotides containing a single basepair mismatch where the top strand was labeled with a FAM fluorophore (blue traces) and the bottom strand is labeled with a ROX fluorophore (red traces). Samples were analyzed by capillary electrophoresis and cleavage products (+M0678 samples) are evidenced by the shifting of the substrate peaks with regards to the non-enzyme treated samples (-M0678) in the T:T, G:T and G:G mismatched substrates. Moreover, some slight cleavage of the T-containing strand (blue strand) of the T:C mismatch can be seen in 30 minutes. This nicking activity on a T:C mismatch substrate increases over time (up to 18 h, data not shown).
Product Source
An engineered mismatch-specific endonuclease expressed in E. coli.
This product is related to the following categories:
DNA Repair Enzymes and Structure-specific Endonucleases Products
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