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Argonaute protein mutant and application thereof

A mutant and protein technology, applied in the field of Argonaute protein mutants and their applications, can solve the problems of time-consuming, limited range of target DNA, complicated operation, etc.

Active Publication Date: 2019-09-13
BERRYGENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention provides an isolated Argonaute (Ago) protein mutant, which has DNA binding activity but lacks DNA cleavage activity, so it can be used for easy-to-operate, efficient and accurate enrichment of target DNA, thereby solving the problem of utilizing existing When technology (especially nucleic acid probe-based hybrid capture method and dCas9-based capture method) enriches the target DNA sequence, the target DNA range is limited, time-consuming, complicated operation, poor efficiency and serious off-target problems

Method used

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  • Argonaute protein mutant and application thereof
  • Argonaute protein mutant and application thereof
  • Argonaute protein mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1: Preparation of the Ago protein mutant of the present invention

[0086] Step 1: Construction of expression vector

[0087] A biotin receptor sequence was connected to the N-terminal of the known amino acid sequence (SEQ ID NO: 1) of the Pyrococcus furiosus Ago protein (PfAgo), and a codon-optimized codon for Escherichia coli (E.coli) was designed and synthesized accordingly the nucleotide sequence. The nucleotide sequence, 6x His-Tag, PfAgo-BAS, IRES, BirA (Escherichia coli biotin ligase) were sequentially cloned into the pET-28a vector with the kanamycin resistance gene to obtain the vector pPFA-1.0.

[0088] Using the Q5Site-Directed Mutagenesis Kit (NEB, Cat#E0554S), the pPFA-1.0 was subjected to site-directed mutagenesis according to the operating procedures in the manual. The DNA obtained after mutation was transformed into E. Coli DH5α cells, and cultured overnight at 37°C in LB agarose medium containing kanamycin. For each mutation, 10 colonie...

Embodiment 2

[0100] Embodiment 2: enrich target DNA according to the method of the present invention

[0101] The target DNA in this example is the exon 18-21 fragment of EGFR gene from free DNA in plasma samples and genomic DNA in leukocytes isolated from normal human peripheral blood, respectively.

[0102] Step 1: Extract DNA

[0103] For free DNA: take 4mL of human plasma, use QIAamp Circulating Nucleic Acid Kit (Qiagen, Cat#55114) to extract free DNA according to the kit instructions, and then elute with 45uL Elution Buffer.

[0104] For genomic DNA: take 200uL of leukocytes isolated from human peripheral blood, use MagJET Whole Blood gDNAKit (ThermoFisher, Cat#K2741), and extract genomic DNA according to the instructions of the kit. About 500 ng (30 uL) of the extracted genomic DNA was sonicated (Sonicator Biorupter Pico from Diagenode SA).

[0105] Step 2: Design Guide DNA (gDNA)

[0106] According to the sequence of EGFR exons 18, 19, 20, and 21, gDNA with 5' phosphorylation m...

Embodiment 3

[0121] Embodiment 3: construct the sequencing library of target DNA according to the method of the present invention

[0122] Step 1: Cell-free DNA extraction

[0123] Take 4mL of human plasma, use QIAamp Circulating Nucleic Acid Kit (Qiagen, Cat#55114) to extract free DNA according to the kit instructions, and finally free DNA is eluted with 45uL Elution Buffer provided by the kit.

[0124] Step 2: Ligation of Sequencing Adapters

[0125] Using the KAPA Hyper Prep Kit (Kapa Biosystems, Cat#KK8501) according to the instructions, the free DNA was filled in at the end and A was added, and then ligated with the TruSeq adapter suitable for the Illumina sequencing platform.

[0126] Step 3: Pre-amplification of Ligation Products

[0127] Prepare the reaction system according to the following table:

[0128]

[0129] On the PCR instrument, perform pre-amplification according to the following conditions:

[0130]

[0131]

[0132] After the amplification was complete, ...

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Abstract

The present invention relates to an Argonaute protein mutant which lacks DNA cleavage activity, but has DNA binding activity. The mutation of the mutant is located in a PIWI domain. The invention alsorelates to application of the protein mutant particularly in the enrichment of target DNA and in the construction of a sequencing library. Therefore, the present invention also relates to a method for enriching the target DNA. The method comprises the steps of: (a) designing a leader sequence for a specific sequence in the target DNA; (b) combining the mutant, the leader sequence and the target DNA according to the present invention to obtain a mutant-leader sequence-target DNA ternary complex; (c) capturing the mutant-leader sequence-target DNA ternary complex through a capture medium; and (d) isolating the target DNA from the captured mutant-leader sequence-target DNA ternary complex to obtain enriched target DNA.

Description

technical field [0001] The present invention relates to a wild-type Argonaute protein (Ago)-based mutant that lacks DNA cleavage activity but has DNA-binding activity, and uses of the protein mutant, especially in enriching target DNA and constructing sequencing libraries . The invention also relates to kits comprising said protein mutants. Background technique [0002] Efficient enrichment of DNA in the target region can effectively reduce the cost of sequencing and increase the depth of sequencing. For applications that usually require high-depth sequencing, such as somatic mutation detection, the performance of target region enrichment is the main factor determining its sensitivity and specificity1. [0003] The current mainstream target region enrichment methods mainly include (1) multiple primer amplification and (2) nucleic acid probe hybridization capture method2. (1) The target region enrichment method based on multiple primer amplification, using tens to thousand...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/10C12Q1/6806C40B50/06
CPCC12N9/22C12N15/1093C12Q1/6806C40B50/06C12Q2525/191C12Q2521/327C40B40/06C12Q2537/159C12Q2565/531C07K14/195C07K2319/85
Inventor 张建光毛爱平
Owner BERRYGENOMICS CO LTD
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