Method for quickly and stably measuring enzyme activity of Tn5 transposase

Abstract

The invention discloses a method for quickly and stably measuring the enzyme activity of Tn5 transposase. The method includes steps of (1), mixing activity measuring substrates, the transposase and reaction buffer solution with one another; (2), carrying out transposition reaction; (3), adding denaturants [such as SDS (sodium dodecyl sulfonate) and guanidine hydrochloride] into reaction products to carry out denaturation on the transposase, and separating the transposase from DNA (deoxyribonucleic acid) fragments; (4), adding media capable of identifying labels of substrates and binding the media with the unreacted substrates; (5), removing the media bound with the substrates by selective means (such as centrifuging, filtering and concentrating); (6), detecting the content of released DNAwith labels, and comparing the content to standard numerical values to obtain the activity of the Tn5 transposase. The method for measuring the enzyme activity has the advantages that the method is easy and convenient to operate and quite suitable for detecting the enzyme activity of the Tn5 transposase during mass production, and is speedy, and results are good in repeatability.

Description

technical field [0001] The invention relates to the technical field of biology, in particular to a fast and stable method for measuring the enzyme activity of Tn5 transposase. Background technique [0002] With the development of sequencing technology, high-throughput sequencing has been widely used in many fields such as genomics, transcriptomics, and medical detection. High-throughput sequencing technology services and related sequencing reagent consumables have become commercialized. An important branch of the biotechnology industry. High-throughput sequencing technology includes three steps: library building, sequencing reaction, and data processing. The library building step processes the DNA to be sequenced to prepare DNA fragments that can be used for sequencing; the sequencing reaction uses the synthesis reaction of DNA polymerase, combined with fluorescence detection technology, Determine the sequence of the DNA fragments; the data processing step compares and asse...

AI Technical Summary

Problems solved by technology

These detection processes all use the transformation method to allow the transposase and the reporter gene to enter the interior of the bacterial cell, which makes the test result (positive plaque) closely related to the transformation efficiency; however, the stability of the transformation experiment itself is poor, and the transformation result is the same as the feeling. The titer of competent cells has a lot to do with it. In the specific experimental operation, even if it is stored at -80°C, it is very difficult to ensure the stability of the titer of competent cells for a long time.

At the same time, due to the bacterial culture involved, the overall activity test reaction time is longer

This makes the determination and comparison of enzyme activity between different production batches a troublesome problem in the production of Tn5 transposase

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