Cascade rolling-ring amplification strategy mediated with dumbbell molecular probe under protection of target object and applied to sensitiveness detection of DNA transmethylase activity

A technology of methyltransferase and rolling circle amplification, which is applied in the direction of recombinant DNA technology, microbial measurement/testing, DNA/RNA fragments, etc., can solve complex probe design, sensitivity reduction, and affect detection accuracy and sensitivity and other issues to achieve the effect of improving the sensitivity

Active Publication Date: 2016-02-24
SHANDONG UNIV
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Problems solved by technology

Although these methods achieve sensitive DNA methyltransferase activity analysis, there are still some limitations that affect the detection accuracy and sensitivity.
The DNA trigger strand in double-stranded DNA or hairpin DNA is in a semi-closed state, so the hybridization of DNA molecular probes and subsequent signal amplification probes or non-specific effects of probes and nucl

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  • Cascade rolling-ring amplification strategy mediated with dumbbell molecular probe under protection of target object and applied to sensitiveness detection of DNA transmethylase activity
  • Cascade rolling-ring amplification strategy mediated with dumbbell molecular probe under protection of target object and applied to sensitiveness detection of DNA transmethylase activity
  • Cascade rolling-ring amplification strategy mediated with dumbbell molecular probe under protection of target object and applied to sensitiveness detection of DNA transmethylase activity

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[0052] Example 1

[0053] Materials and reagents

[0054] All DNA sequences were synthesized and purified by Shanghai Bioengineering Co., Ltd. (Shanghai, China). The methyltransferases M.SssI, HaeIII, AluI, restriction endonucleases HpaII, RsaI, T4 DNA ligase and DNA polymerase were all purchased from New England Biolabs (Ipswitch, MA). Enzyme reaction buffer and S-adenosyl-L-methionine (SAM) are also provided by NewEnglandBiolabs. SybrGreenI was purchased from Biotech (Beijing, China). Methyltransferase inhibitors 5-azacytosine (5-Aza) and 5-aza-2'-deoxycytosine (5-Aza-dC) were purchased from Sigma-Aldrich (St. Louis, MO, USA ). All reagents are of analytical grade, and the water used is ultrapure water (> 18.25MΩcm -1 ).

[0055] Table 1

[0056]

[0057] Equipment

[0058] Fluorescence spectrophotometer (F-7000, Hitachi, Japan); Electrothermal constant stability incubator (DNP-9052, Shanghai Jinghong Experimental Equipment Co., Ltd.); Constant temperature circulating water tank...

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Abstract

The invention provides a cascade rolling-ring amplification strategy mediated with a dumbbell molecular probe under protection of a target object and applied to sensitiveness detection of DNA transmethylase activity. Based on the cascade rolling-ring amplification strategy mediated with the dumbbell molecular probe under protection of the target object, a novel fluorescence detection system for DNA transmethylase activity analysis is developed. The detection system has excellent specificity and sensitivity, the detection limit is as low as 0.0024 U/mL, and potential application value is exhibited in quantitative monitoring of transmethylase activity and screening of anticarcinogen.

Description

technical field [0001] The invention relates to the field of enzyme activity detection, in particular to a method, a probe and a kit for detecting DNA methyltransferase activity by a cascade rolling circle amplification strategy mediated by a target-protected dumbbell molecular probe. Background technique [0002] The function of DNA methyltransferase is to maintain the methylation pattern of the genome, which plays an important role in regulating gene expression and maintaining genome stability. Abnormalities in the activity of methyltransferases lead to abnormal DNA methylation patterns that silence tumor suppressor genes and lead to cancer. Therefore, DNA methyltransferase activity has become a predictive marker and therapeutic target for many cancers. In addition, abnormalities in methyltransferase activity often precede other signs of malignancy and thus have potential applications in the early diagnosis of cancer. Therefore, accurate and sensitive methyltransferase a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/125C12Q2525/131C12Q2563/107
Inventor 姜玮王磊赵海燕
Owner SHANDONG UNIV
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