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
CN104297221AActive Publication Date: 2015-01-21VAZYME BIOTECH NANJING
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
VAZYME BIOTECH NANJING
Publication Date
2015-01-21

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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.
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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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