Ring mediated cascade amplification strategy used for detecting DNA transmethylase activity in high sensitivity manner
A methyltransferase and activity technology, applied in the field of enzyme activity detection, can solve the problems of non-specific background amplification, probe leakage non-specific amplification, affecting detection sensitivity and accuracy, etc. selective effect
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Embodiment 1
[0067] 1. Analysis of Dam methyltransferase activity
[0068] To 10 μL methyltransferase buffer (50mM NaCl, 10mM Tris-HCl, 10mM MgCl 2 , 1mMdithiothreitol, pH 7.5) were added with 160μM SAM (SAM is a cofactor), 20nM long stem-loop probe, 2U DpnI and different amounts of Dam methyltransferase, and incubated at 37°C for 2h. Subsequently, 40nM hairpin probe, 1UKlenow Fragment (3′–5′exo-), 2U Nt.BbvCI and 0.6mM dNTPs and 1×CutSmart (50mM KAc, 20mM Tris-HAc, 10mM Mg( Ac) 2 , 100μg / mL BSA, pH 7.9), incubate at 37°C for 30 min. The reaction system was heated at 85 °C for 20 min to inactivate the enzyme, and then slowly cooled to 30 °C. After that, 120U T4 DNA ligase, 800nM padlock probe and 1×T4 ligase buffer (50mM Tris-HCl, 10mM MgCl 2 , 10mMdithiothreitol, 1mM ATP, pH 7.5), incubated at 37°C for 1h. Then 1mM dNTPs, 3U Phi29 DNA polymerase, 4U Nt.BbvCI and 1×CutSmart were added, and incubated at 37°C for 3h. The reaction system was heated at 75 °C for 20 min to inactivate the ...
Embodiment 2
[0074] 1. Principle analysis
[0075] The proposed rationale for the assay of methyltransferase activity is as follows: figure 1shown. When Dam methyltransferase exists, it specifically recognizes and catalyzes the methylation of long stem-loop probes to form methylated long stem-loops. Subsequently, the DpnI restriction enzyme specifically cuts the methylated long stem-loop into two parts. One part is double-stranded DNA, and the other part is new hairpin probe. Under the experimental conditions, the structure of the new hairpin probe is unstable, and a conformational transition occurs to form a single strand. Under the action of Klenow Fragment polymerase and Nt.BbvCI, the single-stranded DNA initiates polymerization and cleavage reactions, releasing multiple primers. Under the action of T4 DNA ligase, the released primer hybridizes with the padlock probe to obtain a circular probe. Subsequently, this primer triggers rolling circle amplification under the action of Phi2...
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