The type of methylase and its effect on restrictive enzymatic cutting.

-related topics recombination
DNA
technical tool enzymes 1, types of methylasesprimary nuclear biomethylases are used as part of a limitation and modification system to protect host DNA from being cut by the corresponding restriction enzymes. In E.coli, most have three-bit-specific DNA methylase..1. Dam methylaseDam methylase can introduce methyl at the Adenine N6 position in the GATC sequence. Some of the identification bits of the restriction enzymes (PvuII., BamHI., Bcl I., Bgl II., Xho II., MboI., Sau3AI.) contain GATC sequences, while others contain ClaI.(1/4), Part of the identification sequence for XbaI.(1/16), TaqI. (1/16), MboI. (1/16), HphI. (1/16) contains this sequence, such as an average of 4 ClaI. There is one sequence in the bit point (ATCGATN).some restriction enzymes are sensitive to Dam methylated DNA and cannot cut sequences such as BclI., ClaI., XbaI. Insensitive to methylation are BamHI., Sau3AI., BglII., PvuI. and so on. MboI. and Sau3AI. identify and cut bits the same, but the difference is that the former is sensitive to methylation.general mammalian DNA does not methylate on adenine N6, so methylation-sensitive restriction enzyme cutting of these DNAs is not affected. When DNA needs to be fully cut at these sensitive points, it can be amplified and extracted using dam-E.coli..2. Dcm methylase Dcm methylase identifies the CCAGG or CCTGG sequence and introduces methyl at the C5 position of the second cytosine C. Enzymes affected by dcm methylation are EcoRII. (CCWGG). In most cases, this effect can be avoided by its isolytic enzyme BstNI. (CC-WGG) because the identification sequences are the same, but the cut points are different.enzymes affected by this methylase are Acc65I. , AlwNI., ApaI., EcoRII. and EaeI. Enzymes not affected by this methylation are BanII., Bg1I., BstNI., KpnI. and NarI..3.EcoKI. Methylase EcoKI. Methylase has fewer identification points and identifies the N6 position of A in the AAC(N) 6GTGC and GCAC (N) 6GTT sequences. However, because there are fewer identifying bits (1/8kb), there is less research..4.SsSI. Methylase SssI. Methylase comes from the original nuclear organism Spiroplasma, which enables C in the CG sequence to be methylated at the C5 position. Methylation templates can be DNA strands of methylated or semi-methylated chains (new synthetic chains). SsSI. Methylated DNA is limited by the mcrA, mcrBC, and mrr systems in E.coli. Many enzymes are sensitive to this methylation, such as AatII., ClaI., XhoI., Sal I. etc., and are also insensitive, such as BamHI., EcoRI., Sph I. and KpnI.2. There are at least three methylation-dependent restriction systems E.coli, mcrA, mcrBC, and mrr, which identify different sequences, but identify only methylated sequences, all of which limit DNA (restrictions, i.e. digestion and degradation) acting by CpG methylase (M SssI). Mrr limit m6A; McRA limits the bits of HpaII. methylation modification; McRBC cuts two sets of half-bits (G/A)mC, which are 2kb apart, most 55 to 103bp and require GTP;, the effect of methylation on limiting enzymatic cutting 1. modified enzyme cut points HincII. Four bits can be identified (GTCGAC, GTCAAC, GTTGAC and GTTAAC), methylase M.TaqI. A in methylated TCGA, so M.TaqI. after DNA treatment, GTCGAC will not be subject to GTCGAI.. The product of the M.MspI. modification is m5CCGG, and if it is CC or GG in front of the BamHI. Identification Point (GGATCC), then M.Msp. processed DNA (GGAT m5CCGG) is insensitive to BamHI. (i.e., resistance to cutting). the DNA library, when the DNA library is partially digested with AluI.
(AG-CT) and HaeIII.(GG-CC), the
resulting fragments are treated with M.EcoRI. methylase, followed by synthetic EcoRI. connectors, and then cut with EcoRI. only the bits on the joints can be cut to protect the
genome
fragments. . 2. The production of new enzyme cut can be generated by methylation modification of the new enzyme cutting point. DpnI. is a restriction enzyme that relies on methylation, and TCGATCGA is treated by M.TaqI. to form methylation (A) product TCG-ATCG-A, where G-ATC is the DpnI. bit. . 3. Effects on genomic mapping In studying methylation levels and distributions of mammalian m5CG, plant m5CG and m5CNG, and intestinal cells Gm6ATC, the sensitivity of limiting enzymes to methylation varies greatly.
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