Mismatch Endonuclease I (M0678S)는 Mg2+ dependent DNA endonuclease로 mismatched base pairs(아래 참조)를 특이적으로 절단합니다.
- 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
- 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
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.