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Active determination method for Lambda exonuclease

An exonuclease and activity determination technology, applied in the field of biochemistry, can solve the problems of radioactive pollution, difficulty in achieving high throughput, automation, and many steps, and achieve the effect of simple and fast operation steps, automatic activity determination, and high sensitivity

Active Publication Date: 2015-01-21
VAZYME BIOTECH NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is prone to radioactive contamination, and has many steps and a long cycle, making it difficult to achieve high-throughput and automation

Method used

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  • Active determination method for Lambda exonuclease
  • Active determination method for Lambda exonuclease
  • Active determination method for Lambda exonuclease

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0018] 1. Preparation of Phosphorylated Double-Stranded DNA

[0019] Primer F (5' phosphorylation modification): 5'-aataacgtcggcaactttgg-3';

[0020] Primer R: 5'-gttacgccaccagtcatcct-3';

[0021] PCR template: λ DNA

[0022] PCR reaction:

[0023] components volume DDW to 50μl 10x Taq buffer 5 dNTPs (10 mM) 1μl Primer F (10 μM) 2μl Primer R (10 μM) 2μl lambda DNA 10ng Taq enzyme 1 U

[0024]

[0025] The PCR product was extracted with phenol-chloroform and purified by ethanol precipitation. The concentration of the purified PCR product was determined by A260.

[0026] 3. Lambda exonuclease reaction:

[0027] components volume DDW to 50μl 10 x λ exonuclease buffer 5 Phosphorylated PCR product 100ng lambda exonuclease 1 mU-1024 mU

[0028] temperature time 37℃ 30 minutes 4℃ Keep

[0029] 4. Picogreen fluorescence detection:

[0030] Ad...

Embodiment 1

[0034] Embodiment 1. Establishment of the method for measuring lambda exonuclease activity by fluorescence method

[0035] 1. Preparation of Phosphorylated Double-Stranded DNA

[0036] Primer F (5' phosphorylation modification): 5'-aataacgtcggcaactttgg-3';

[0037] Primer R: 5'-gttacgccaccagtcatcct-3';

[0038] PCR template: λ DNA

[0039] PCR reaction:

[0040] components volume DDW to 50μl 10x Taq buffer 5 dNTPs (10 mM) 1μl Primer F (10 μM) 2μl Primer R (10 μM) 2μl lambda DNA 10ng Taq enzyme 1 U

[0041]

[0042] The PCR product was extracted with phenol-chloroform and purified by ethanol precipitation. The concentration of the purified PCR product was determined by A260.

[0043] 3. Lambda exonuclease reaction:

[0044] components volume DDW to 50μl 10x lambda exonuclease buffer 5 Phosphorylated PCR product 100ng Lambda exonuclease 0-1024 mU

[0045] ...

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Abstract

The invention discloses an active determination method for Lambda exonuclease. The active determination method comprises the following steps: obtaining double-chain DNA with a phosphorylated 5' end on one chain, then reacting in a reaction system in the presence of the Lambda exonuclease by adopting the double-chain DNA as a substrate, determining the relative quantity of single-chain DNA or the double-chain DNA in the reaction system after the reaction is finished, and deducing the active degree of the Lambda exonuclease from a detection result. Compared with the method in the prior art, the active determination method has the advantages of no radioactive pollution, simplicity and fastness in operation and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a method for measuring lambda exonuclease activity. Background technique [0002] The lambda exonuclease is encoded by the lambda phage exonuclease gene. λ exonuclease acts on double-stranded DNA and cuts off 5' mononucleotide step by step along the 5'→3' direction. The optimal substrate is 5' phosphorylated double-stranded DNA. [0003] Lambda exonuclease is an important tool enzyme in genetic engineering technology, which can be used to prepare single-stranded DNA templates. The standard bioassay method adopts the radioisotope method, that is, a 5' terminal band is first synthesized 32 P-labeled dATP double-stranded DNA. The lambda exonuclease activity converts [ 32 P]-dATP excision. After TCA precipitation and whatman filter paper filtration, the λ exonuclease activity was calculated by measuring the radioactive content in the acid-soluble substances. Th...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 徐晓昱
Owner VAZYME BIOTECH NANJING
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