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A technology for glycosylase activity and determination method, which is applied in the field of uracil-DNA glycosylase activity determination, can solve the problems of radioactive pollution, many steps, difficulty in achieving high-throughput, automation, etc., and achieve simple and fast operation steps , the effect of high sensitivity
Inactive Publication Date: 2015-01-21
VAZYME BIOTECH NANJING
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This method is prone to radioactive contamination, and has many steps a...
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[0020] 1. Preparation of UDNA
[0021] Primer F: 5'-cggatcgccaacactcacaacaat-3';
[0022] Primer R: 5'-aaaggccgggaaatacccagcc-3';
[0023] PCR template: λ DNA
[0024] PCR reaction:
[0025] components volume DDW to 50μl 10x Taq buffer 5 dU / A / G / CTP mixture 1μl Primer F (10 μM) 2μl Primer R (10 μM) 2μl lambda DNA 10ng Taq enzyme 1 U
[0026]
[0027] The PCR product was extracted with phenol-chloroform and purified by ethanol precipitation. The concentration of the purified PCR product was determined by A260.
[0028] 2. UDG response:
[0029] components volume DDW to 50μl 10 x Taq buffer 5 UDNA 100ng UDG 1mU-1024mU temperature time 25℃ 30 minutes 4℃ Keep
[0030] 3. Picogreen fluorescence detection:
[0031] Add 0.5 μl of picogreen dye (invitrogen P11495) to the reaction product; detect with a fluorescent microplate reader, the excitation wavel...
Embodiment 1
[0035] Embodiment 1. Establishment of the method for measuring UNG activity by fluorescence method
[0036] 1. Preparation of UDNA
[0037] Primer F: 5'-cggatcgccaacactcacaacaat-3';
[0038] Primer R: 5'-aaaggccgggaaatacccagcc-3';
[0039] PCR template: λ DNA
[0040] PCR reaction:
[0041] components volume DDW to 50μl 10x Taq buffer 5 dU / A / G / CTP mix 1μl Primer F (10 μM) 2μl Primer R (10 μM) 2μl lambda DNA 10ng Taq enzyme 1 U
[0042]
[0043] The PCR product was extracted with phenol-chloroform and purified by ethanol precipitation. The concentration of the purified PCR product was determined by A260.
[0044] 2. UDG response:
[0045] UDG for NEBM0280L
[0046] components volume DDW to 50μl 10 x Taq buffer 5 UDNA 100ng UDG 1mU-1024mU
[0047] temperature time 25℃ 30 minutes 4℃ Keep
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Abstract
The invention discloses a uracil-DNA glycosylase activity measurement method. The method comprises the following steps: firstly, carrying out PCR amplified reaction; with a double-chain DNA as a template, carrying out polymerization reaction by using polymerase in the presence of a dU/A/G/CTP mixture and a primer, and carrying out amplification to generate amplification product UDNA double chains containing dU; secondly, carrying out uracil-DNA glycosylase reaction, and forming a lot of DNA chains in base deletion from the amplification product through the uracil-DNA glycosylase action; and finally, measuring the relative quantity of a single-chain DNA product or UDNA double chains in the reaction system, and deriving the uracil-DNA glycosylase activity degree according to the measurement result, wherein the uracil-DNA glycosylase activity degree is inversely proportional to the quantity of the DUNA double chains in the reaction system, and is directly proportional to the quantity of the single-chain DNA product. The method disclosed by the invention is free of radioactive pollution, simple and rapid in operation, high in sensitivity, and stable, and quantitative determination can be carried out.
Description
technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a method for measuring the activity of uracil-DNA glycosylase. Background technique [0002] UDG enzyme, Uracil-DNA Glycocasylase (Uracil-DNA Glycocasylase). The principle of action of UDG is to selectively hydrolyze and break the uracil glycosidic bond in double-stranded or single-stranded DNA containing dU to form a DNA chain with missing bases, which will be further hydrolyzed and broken in alkaline medium and high temperature, thereby being eliminated. UDG is an important component of the DNA damage repair system in cells, and it exists widely in nature, including bacteria, eukaryotes, and human cells that express UDG enzymes. [0003] UDG plays a very important role in PCR anti-pollution. The most common and most important contaminant in PCR reactions is PCR amplification product contamination. Because the sensitivity of PCR is very high, a single copy ca...
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