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Method for identifying open chromatin sites of plant genomes by using micrococcal nuclease

A technology of micrococcal nuclease and plant genome, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem of not being able to directly identify the core sequence of functional DNA elements, limited application of plant genome, and increased experimental costs, etc. problem, to achieve strong visualization effect, short time-consuming, and easy-to-repeat effect

Active Publication Date: 2019-05-03
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ATAC-seq is currently widely used in human and animal genomes, but its application in plant genomes is also very limited, mainly because the method requires very high uniformity of plant cells, and plant tissues are usually composed of many cell types The composition of the mixture requires the use of flow cytometry to sort the cells to ensure the homogeneity of the cells, thus limiting the application of this method in laboratories with limited experimental conditions
At the same time, the success rate of this method is low, which increases the experimental cost
On the other hand, none of the above three methods can be used to directly identify the core sequence of a functional DNA element

Method used

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  • Method for identifying open chromatin sites of plant genomes by using micrococcal nuclease
  • Method for identifying open chromatin sites of plant genomes by using micrococcal nuclease
  • Method for identifying open chromatin sites of plant genomes by using micrococcal nuclease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] (1) Plant material cross-linking:

[0063] Select 2-5g of the experimental material (wheat, corn, rice or Arabidopsis leaves) to be tested, cut into pieces of a certain size, soak in the final concentration of cross-linking fixative, and cross-link at 4°C for 10 minutes under vacuum conditions .

[0064] A 2.5M glycine solution was added to the cross-linking liquid to obtain a final concentration of 125 mM, and vacuum treatment was performed at 4° C. for 5 minutes.

[0065] Discard all the cross-linking solution, rinse three times with sterilized distilled water, place all the leaves on absorbent paper, and dry them in the air for 10 minutes. Wrapped in paper and frozen in liquid nitrogen for 10 minutes, the leaves were taken out and stored in a -80°C refrigerator for later use.

[0066] (2) extracting and purifying the nucleus of the plant material:

[0067] The fixed leaves were ground into powder with liquid nitrogen, and 10 ml of the powder was taken to extract cel...

Embodiment 2

[0083] Example 2 RE-MNase-seq identification of open chromatin loci in wheat genome

[0084] Select 60U MNase enzyme-digested wheat cell nuclear DNA, separate by 1.5% agarose gel electrophoresis, and then reclaim all DNA fragments that do not contain 150bp in 50-280bp for construction of Illumina sequencing library ( Figure 7 ), separation and purification of 200-450bp DNA fragments ( Figure 8 ) for 2x150PE sequencing on the Illumina Nova-seq sequencing platform.

[0085] Quality control analysis was performed on the sequencing data, and the results are shown in Table 2. The results of preliminary quality analysis showed that the alignment rate of the whole wheat genome was 97.32%, and that of Q20 was 64%-69%, indicating that the data quality was reliable and could be used for the identification and functional analysis of open chromatin loci in the whole wheat genome. A total of 100,000 sites were initially identified ( Figure 9 ), through correlation analysis with gene ...

Embodiment 3

[0088] Example 3 RE-MNase-seq identifies open chromatin loci in the maize genome.

[0089] Select 60U MNase enzyme-digested corn nuclear DNA, separate it by 1.5% agarose gel electrophoresis, and then recover all DNA fragments excluding 150bp in 50-280bp for construction of Illumina sequencing library, separate and purify 200-450bp DNA fragments for Illumina Nova- The seq sequencing platform performs 2x150PE sequencing.

[0090] Quality control analysis was performed on the sequencing data. The preliminary quality analysis results showed that the comparison rate of the whole maize genome was 94.50%, and the comparison rate of Q20 was 45%, indicating that the data quality is reliable and can be used for the identification of open chromatin sites in the whole maize genome. Identification and functional analysis. A total of 40,000 loci were initially identified ( Figure 13 ), through the association analysis of the relationship with gene expression, it was found that at the gen...

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Abstract

The present invention discloses a method for identifying open chromatin sites of plant genomes by using micrococcal nuclease. The method comprises the following steps: (1) cross-linking plant material; (2) extracting and purifying cell nucleus of a plant material; (3) subjecting the cell nucleus to enzyme digestion with MNase in different concentrations; (4) detecting MNase digestion effect by 1.5% agarose gel electrophoresis; (5) selecting appropriate MNase for enzyme digestion of nuclear DNA, performing 1.5% agarose gel electrophoresis separation again, recovering DNA fragments, constructinga Illumina sequencing library, and performing sequencing; and (6) performing bioinformatics analysis on the sequencing data, and identifying the whole genome MH loci. The whole method is simple in process and short in time consuming, takes about 2.5 days in one cycle, and is high in visible enzyme digestion effect and suitable for most plant species. The method can be used directly used for identifying the core regions of functional DNA elements.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a new method for identifying open chromatin sites in plant genomes by using micrococcal nuclease. Background technique [0002] In animal and plant genomes, genome-wide chromatin can be divided into two categories according to the chromatin structure: one is open chromatin, where the abundance of nucleosomes is low or even no nucleosome structure exists; the other is open chromatin. Closed chromatin, a region with prominent or high abundance nucleosomes present. Compared with closed chromatin, open chromatin regions usually contain functional genes or functional DNA regulatory elements in a series of different expression states, such as promoters and enhancers. Open chromatin plays a very important regulatory role in the normal growth and development of eukaryotes, the differentiation of tissues and organs, and the response to internal and external environmental factors....

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
Inventor 张文利林堪德郑东洋陶申童冯逸龙陈莉芬张鹏越
Owner NANJING AGRICULTURAL UNIVERSITY
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