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Mismatch Endonuclease I

Catalog # Concentration Size List Price Quantity Your Price
M0678S 80000 units/ml 4000 units $235.00
$211.50
Catalog # Size List Price Your Price
M0678S 4000 units $235.00
$211.50
Catalog #
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  • DNA Endonuclease
  • Catalyzes the cleavage of some DNA mismatches (T:T, G:G and G:T)
  • Cleaves the 3rd phosphodiester bond on the 5′ side of the mismatched base in both strands leaving a 5 bp overhang with a 3′- OH and 5′- phosphate
  • Best at T mismatches; does not recognize all DNA mismatches   
 
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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.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  Mismatch Endonuclease I M0678SVIAL -20 1 x 0.05 ml 80,000 units/ml
  NEBuffer™ r2.1 B6002SVIAL -20 1 x 1.25 ml 10 X
Features
  • Cleaves T:T, G:G and T:G mismatches in DNA
  • Cleaves T:I, G:I and G:U mismatches in DNA
  • Cleaves T:G and T:U (DNA:RNA) mismatches
  • Nicks thymine strand of T:C mismatches in DNA
 
An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

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Notes
  • One unit is defined as the amount of enzyme required to cleave 50% of 0.2 pmol of a single T:T mismatch in 30 minutes at 37°C in a total reaction volume of 20 µl in 1X NEBuffer r2.1. 1 µl of Mismatch Endonclease I contains 80 U, therefore, 1 µl of Mismatch Endonuclease I is able to completely cleave 8 pmol of mismatched DNA.
  • The addition of molecular crowding agents (PEG, non-specific DNA) and some detergents (Triton X-100) may increase the relative activity of Mismatch Endonuclease I on some mismatches.
Citations
Additional Citations
  • Baljinnyam, T., Conrad, J., Sowers, M., Chang-Gu, B., Herring, J., Hackfeld, L., Zhang, K. and Sowers, L. (2022) Characterization of a Novel Thermostable DNA Lyase User to Prepare DNA for Next-Generation Sequencing. Anal Chem DOI: 10.1021/acs.chemrestox.2c00172
Quality Control Assay
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Specifications
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Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Safety Data Sheets
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