Fluorescent biosensor for detecting DNA methylation, and detection method and application thereof
A biosensor, methylation technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc., can solve the problems of complex reaction steps, limited wide application, time-consuming and labor-intensive pretreatment process, etc. The method is simple, the experimental scheme is simplified, and the sensitivity is improved.
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[0059] Detection of DNA methylation:
[0060] Drugs and materials: All oligonucleotides were synthesized and purified by HPLC at Sangon Bioengineering Co., Ltd. (Shanghai, China). Restriction enzyme HpaII (HpaII), 10×Cutsmart buffer (500 mmol potassium acetate (KAc), 200 mmol tris(hydroxymethyl)aminomethane-acetic acid (Tris-Ac), 100 mmol magnesium acetate ( Mg(Ac) 2 ), 100 micrograms per milliliter bovine serum albumin (BSA), pH 7.9), endonuclease IV (Endo IV), 10×NEB buffer 3 (1 mole sodium chloride (NaCl), 500 mmol tris(hydroxy Methyl)aminomethane-hydrochloric acid (Tris-HCl), 100 mmol magnesium chloride (MgCl 2 ), 10 mmol dithiothreitol (DTT, pH 7.9) and NEBNext DNA double-strand fragmentation enzyme (NEBNext dsDNA Fragmentase) were purchased from New England Bio (Ipswich, MA, USA). Streptavidin-coupled superparamagnetic beads ( M-280 Streptavidin) was purchased from Thermo Fisher Corporation (Carlsbad, CA, USA). All other reagents were of analytical grade and used w...
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