DNase I mutants as well as coding nucleotide sequences and application thereof

A mutant and sequence technology, applied in the field of DNaseI mutants, can solve the problems of large loss, unstable effect of micro-sample interruption, and sequencing quality needs to be improved, so as to achieve small sample loss, excellent sequencing quality, and low GC preference. Effect

Active Publication Date: 2021-08-06
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Relevant literature and related tests at this stage show that although DNase I is simple and easy to apply to the preparation of high-throughput sequencing libraries, there are still some disadvantages, that is, the interrupting effect of micro-sample is not stable enough, and the loss is relatively large; preference, the quality of sequencing needs to be improved

Method used

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  • DNase I mutants as well as coding nucleotide sequences and application thereof
  • DNase I mutants as well as coding nucleotide sequences and application thereof
  • DNase I mutants as well as coding nucleotide sequences and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 is tested to the different enzyme amount of mutant 7

[0037] 1. Genome disruption

[0038] In this example, 500 ng of calf thymus gDNA (Yeasen, Cat#60612ES03) was used as the fragmentation template, and 5 μL of 10×Fragment buffer was added. 50 μL. Place it in a PCR instrument for the interruption reaction, set the enzyme cutting temperature to 30°C, and the inactivation temperature to 80°C.

[0039] PCR program

[0040] temperature time 4℃ 1 minute 30℃ 15 minutes 80℃ 10 minutes

[0041] 2. Interrupt result detection

[0042] After the reaction program was finished, 15 μL was taken for agarose gel electrophoresis detection. Interrupt results see attached figure 1 .

[0043] 3. Interrupt result analysis

[0044] 1) As the amount of enzyme increases, the interrupted fragments gradually decrease

[0045] 2) 0.6 μL of enzyme can interrupt the genome to about 300bp of the main band.

Embodiment 2

[0046] Example 2 Interruption test of mutant 7 with different input amounts of DNA

[0047] 1. Genome disruption

[0048] In this example, 50, 500, and 1,000 ng of calf thymus gDNA were used as fragmentation templates, and 5 μL of 10×Fragment buffer was added. For mutants, 0.6 μL of enzyme was added, and water was added to 50 μL. Place it in a PCR instrument for interruption, set the enzyme cutting temperature to 30°C, and the inactivation temperature to 80°C.

[0049] PCR program

[0050] temperature time 4℃ 1 minute 30℃ 15 minutes 80℃ 10 minutes

[0051] 2. Interrupt product purification

[0052] In the example, the fragmented product was passed through Hieff NGS TM DNA Selection Beads magnetic beads (Yeaen, Cat#12601) were purified and recovered according to the reagent instructions at a magnetic bead ratio of 1.5× to obtain the fragmented product.

[0053] 3. Interrupt the result of 2100 detection

[0054] The fragmented purified pr...

Embodiment 3

[0057] Example 3 Different interruption time tests for mutant 7

[0058] 1. Genome disruption

[0059] In this example, 500 ng of calf thymus gDNA was used as a fragmentation template, and 5 μL of 10×Fragment buffer was added. The amount of enzyme added to mutant 7 was 0.6 μL, and water was added to 50 μL to prepare 5 groups. The enzyme digestion time was set to 10 and 15 respectively. , 20, 25, 30 min. Place it in a PCR instrument for interruption, the nuclease cutting temperature is 30°C, and the inactivation temperature is 80°C.

[0060] PCR program

[0061] temperature time 4℃ 1 minute 30℃ 10 / 15 / 20 / 25 / 30 min 80℃ 10 minutes

[0062] 2. Interrupt result detection

[0063] In the example, the fragmented product was passed through Hieff NGS TM DNA Selection Beads magnetic beads were purified and recovered at a ratio of 1.5× magnetic beads according to the reagent instructions to obtain the fragmentation product.

[0064] 3. Interrupt th...

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Abstract

According to the invention, wild type DNase I related sites are mutated and screened to obtain three mutants with good effects, wherein two mutants are in double-site mutation, and mutation sites are respectively A249T, R42H and A249T, F110V; one mutant is in three-site mutation, and the mutation sites are A249T, R42H and F110V. The DNase I mutant with the three-site mutation has the best effect, and the DNase I mutant has an amino acid sequence shown in SEQ ID No.1 and a coding nucleotide sequence shown in SEQ ID No.2. The invention also discloses application of the DNase I mutants in construction of a high-throughput sequencing library. The DNase I mutants disclosed by the invention can efficiently and stably break genome DNA, fragmented products of the DNase I mutants can be applied to construction of a high-throughput sequencing library, and compared with wild type DNase I, library construction and sequencing quality is more excellent, and GC preference is lower. Low-cost and high-efficiency library building can be realized, and sequencing quality is excellent.

Description

technical field [0001] The patent of the invention relates to a DNase I mutant, its coding nucleotide sequence and its application, and belongs to the field of biotechnology. Background technique [0002] DNase I, namely Deoxyribonuclease I, the Chinese name is deoxyribonuclease I, is an endonuclease that can digest single-stranded or double-stranded DNA to produce single deoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides . DNase I activity depends on calcium ions and can be activated by magnesium ions or divalent manganese ions. In the presence of magnesium ions, DNase I can randomly cut any position of double-stranded DNA; in the presence of divalent manganese ions, DNase I can cut DNA double-strands at the same position to form blunt ends, or 1-2 Sticky ends with nucleotide overhangs. Using this feature, genomic DNA can be randomly interrupted, and the interrupted products can be applied to high-throughput sequencing library preparation. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/55C40B50/06
CPCC12N9/22C40B50/06C12Y301/11001
Inventor 秦雪梅柴常升曹振宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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