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Transposase activity determination method

A technology of activity measurement and measurement method, applied in the field of transposase activity measurement, can solve the problems of lack and difficult control of enzyme activity measurement, and achieve the effect of short time measurement of activity

Pending Publication Date: 2022-03-29
VAZYME BIOTECH NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is a lack of unified standards and scientific methods for the determination of transposase activity and functional evaluation, which makes it difficult to control the determination and comparison of enzyme activity between different batches in the production of Tn5 transposase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Substrate Construction Containing Transposase Recognition Sequence

[0068] 1. Synthesis of upstream substrates

[0069] Upstream substrate structure: A sequence-ME sequence (19bp)

[0070] Using the C57BL / 6 mouse genome as a template and F3 / R4 as primers, use Nanjing Novizyme Biotechnology Co., Ltd. Cat. No. P515 reagent to synthesize upstream substrates, and use Nanjing Novizyme Co., Ltd. Cat. No. DC301 kit Gel recovery was performed on the upstream substrate.

[0071] F3: 5'-GTTTCTCCTACTCAGCCAGTGGCAC-3'

[0072] R4: 5'-AGATGTGTATAAAGAGACAGTGCACATGGTCGCGTCAGTCC-3'

[0073] 2. Synthesis of Downstream Substrates

[0074] Downstream substrate structure: ME sequence (19bp)-B sequence

[0075] Using the 293T cell genome as a template and F4 / R3 as primers, use Nanjing Novizyme Biotechnology Co., Ltd. Cat. No. P515 reagent to synthesize downstream substrates, and use Nanjing Novizyme Co., Ltd. Cat. for glue recovery.

[0076] F4: 5'-AGATGTGTATAAGAGACAGGCTGA...

Embodiment 2

[0091] Embodiment 2: Transposase activity test

[0092] The transposase activity after treatment at 37°C for 0min / 30min / 2h was tested respectively.

[0093] 1. The transposase is combined with the complete substrate finally obtained in Example 1, and the system is as follows:

[0094] Reagent volume Transposase (500ng / μl) 1μl Intact substrate 50ng / μl 1μl CouplingBuffer 18μl total capacity 20μl

[0095] Transposase and Coupling Buffer were from Nanjing Novizyme Biotechnology Co., Ltd., Cat. No. S601.

[0096] Also take a portion of the complete substrate 50ng finally obtained in Example 1, as a control, the system is as follows:

[0097] Reagent volume Transposase (500ng / μl) 0μl Intact substrate 50ng / μl 1μl CouplingBuffer 19μl total capacity 20μl

[0098] 2. Carry out the interruption reaction, 30°C for 60min.

[0099] 3. Take the interrupted product, dilute it 500 times with sterile wat...

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Abstract

The present invention relates to a transposase activity determination method, particularly to a Tn5 transposase activity determination method, which comprises: combining a transposase with a substrate containing a transposase recognition sequence, carrying out a breaking reaction, carrying out qPCR detection on the broken product, and evaluating the transposase activity according to a Ct value. According to the method, the activity of the transposase can be accurately measured, so that the activity of the transposase is evaluated, and the activity measuring time required by the method is short.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for measuring transposase activity. Background technique [0002] At present, the library construction process of high-throughput sequencing technology can be divided into two methods according to the different interruption methods, namely physical method and enzymatic method. The physical method includes nebulization and ultrasound to interrupt DNA, and the enzymatic method includes DNA fragmentation enzyme. (Fragmentase), DNA shearing enzyme (Shearase) and transposase (Tn5 Transposase), etc., except transposase, all other library construction methods include several steps of breaking DNA, repairing DNA fragments and adding adapters to DNA fragments . When using transposase to build a library, the ultra-high activity Tn5 transposase can perform non-specific random cutting of DNA molecules, and only one step can complete the process of breaking DNA and adding adapters to DN...

Claims

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Application Information

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IPC IPC(8): C12Q1/48C12Q1/6851C12N15/11
CPCC12Q1/48C12Q1/6851G01N2333/91245C12Q2531/113C12Q2563/107
Inventor 瞿志鹏江明扬唐秋雨易文洋
Owner VAZYME BIOTECH NANJING
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