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Mutant Tn5 transposase and kit

A transposase and mutant technology, applied in transferase, library creation, biochemical equipment and methods, etc., can solve the problems of reduced sequencing coverage, low reaction efficiency of Tn5 transposase, loss of target products, etc., to achieve Improved uniformity and coverage, reduced preference, and efficient library construction

Active Publication Date: 2022-07-29
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the wild-type Tn5 transposase has low reaction efficiency and significant bias, which will cause a large loss of target products and reduce sequencing coverage during high-throughput sequencing library construction.

Method used

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  • Mutant Tn5 transposase and kit
  • Mutant Tn5 transposase and kit
  • Mutant Tn5 transposase and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Obtainment of mutant Tn5 transposase

[0027] Wild-type Tn5 transposase has low reaction efficiency and has a significant preference, which will cause a large number of target product losses during high-throughput sequencing library construction and reduce sequencing coverage.

[0028] The present application carries out site mutation on the basis of wild-type Tn5 transposase, and the amino acid mutation sites are: R26W, E54K, R62P, D156K and L372P, and S19R and / or N204K and / or S267T. The amino acid sequence of the wild-type Tn5 transposase is shown in SEQ ID NO: 1. We entrusted Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the coding DNAs of mutants 1-8 and wild-type Tn5 transposase shown in Table 1. Protein expression, mutant Tn5 transposase 1-8 and wild-type Tn5 transposase were obtained. The amount of Tn5 transposase required for complete fragmentation of 1 μg of calf gDNA in 1 h at 55°C was defined as one enzyme activity unit (U).

[0029] ...

Embodiment 2

[0033] Example 2: Application of mutant Tn5 transposase 8 in RNA library construction

[0034] In this example, the RNA extracted from the 293 cell line (purchased from Wuhan Proceeds Life Technology Co., Ltd.) was used for the analysis of the library construction process as shown below.

[0035] First perform RNA denaturation treatment, the denaturation system and denaturation conditions are shown in Table 2; then synthesize the first-strand cDNA, and the synthesis conditions are shown in Table 3; then refer to Yisheng Biotechnology (Shanghai) Co., Ltd. Fast TagmentDNALibrary Prep Kit for (Cat#12207) library construction kit (hereinafter referred to as: 12207 library construction kit) instructions for total RNA construction. Use the mutant 8 described in Example 1, that is, mutant Tn5 transposase 8 and wild-type Tn5 transposase for library construction test, and the rapid RNA library construction process refers to figure 1 ,details as follows:

[0036] 1) Take 0.1ng, 1ng...

Embodiment 3

[0060] Example 3: Application of mutant Tn5 transposase 8 in DNA / RNA co-construction library

[0061] In this example, RNA and calf gDNA (195129-Deoxyribonucleic acid,sodium salt,from calf thymus-50mg-MP-1) extracted from 293 cell line total 293 cell line (purchased from Wuhan Prosser Life Technology Co., Ltd.) Carry out the analysis of the library construction process as shown below.

[0062] First perform RNA denaturation treatment, the denaturation system and denaturation conditions are shown in Table 9; then synthesize the first-strand cDNA, and the synthesis conditions are shown in Table 10; then refer to Hieff of Yisheng Biotechnology (Shanghai) Co., Ltd. Fast Tagment DNALibrary Prep Kit for (Cat.12207) The instructions of the library construction kit were used to conduct DNA / RNA co-construction library. Using the mutant Tn5 transposase 8 and wild-type Tn5 transposase described in Example 1, a rapid DNA / RNA co-construction library was performed, and the library const...

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Abstract

The invention provides a mutant Tn5 transposase and a kit, and belongs to the technical field of biology. On the basis of a wild type Tn5 transposase, the wild type Tn5 transposase has five amino acid site mutations: R26W, E54K, R62P, D156K and L372P, and at least two of three mutation sites: S19R, N204K and S267T, and the amino acid sequence of the wild type Tn5 transposase is as shown in SEQ ID NO: 1. The invention also relates to an application of the mutant Tn5 transposase in library construction. Compared with a wild type Tn5 enzyme, the mutant Tn5 transposase provided by the invention has the advantages that the activity is obviously improved, the preference on a DNA sequence is obviously reduced, dsDNA and DNA / RNA hybrid chains can be recognized without preference, any (0.1-50ng) input amount can be used for building a library within about 2.5 hours, and the requirements of various high-throughput sequencing library building can be met.

Description

technical field [0001] The invention relates to a mutant Tn5 transposase and a kit, belonging to the field of biotechnology. Background technique [0002] With the development of life science, we need to analyze the genetic information of multiple species. NGS, also known as high-throughput sequencing, can read the sequences of hundreds of thousands to millions of DNA molecules at one time, and can provide rich genetic information. The first step in NGS is library preparation, which is critical to the NGS workflow. This step produces a DNA or RNA sample that is compatible with the sequencer. DNA is usually fragmented first, and then specific adapters are added to both ends to construct a sequencing library. [0003] At present, the methods of DNA fragmentation are mainly divided into physical fragmentation or enzymatic fragmentation. The enzyme cleavage method is increasingly becoming the preferred method for NGS library construction because of its high efficiency, conven...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12Q1/6806C12N15/10C40B50/06
CPCC12N9/1241C12Q1/6806C12N15/1096C40B50/06C12Q2521/107C12Q2525/191C12Q2531/113Y02A50/30
Inventor 宋东亮杨春玲刘娜曹振
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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