Monomolecular mechanics method for measuring accurate length of human telomere

A technology of telomere length and molecular mechanics, which is applied in the field of measuring the precise length of human telomere using single-molecule force spectroscopy, can solve problems that have not been reported

Pending Publication Date: 2020-08-18
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, no method for measuring the precise length of human telomeres based on single-molecule force spectroscopy has been reported

Method used

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  • Monomolecular mechanics method for measuring accurate length of human telomere
  • Monomolecular mechanics method for measuring accurate length of human telomere
  • Monomolecular mechanics method for measuring accurate length of human telomere

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Collection of human chronic myeloid leukemia cells K562 cells, extraction of cell genome

[0048] Collection of 1K562 cells

[0049] 1.1 Recovery and replacement of K562 cells

[0050] Take out 1 tube of cells (1.5ml, about 5×10 6 cells) were quickly thawed, and 2ml of complete medium was added (RPMI medium added with a final concentration of 10% fetal bovine serum, 100U / ml penicillin and 100mg / ml streptomycin solution). Centrifuge at 700 rpm for 5 minutes at room temperature, discard the supernatant, and add 5ml of complete medium to resuspend. Then the cells were transferred to 60mm Petri dishes and placed at 37°C, 5% CO 2 in the incubator. Cells attach and proliferate. After 24 hours of static culture, the medium was changed once. Take out the cell culture dish, place it in a biological safety cabinet, discard the medium supernatant, take 5ml of complete medium and slowly add it along the side wall of the culture dish. Continue culturing for 24 hours...

Embodiment 2

[0055] Example 2 Restriction endonuclease combinatorial digestion of genome, retention of telomeric DNA

[0056] The recovered genome was digested with restriction endonucleases (CviAII / NdeI / MseI / BfaI). Take 20 μg of genomic DNA in a 0.2ml PCR tube, add 5 μl of 10×Cutsmart buffer, 10 units of restriction endonuclease CviAII (product number: R0640L, NEB), make up to 50 μl with deionized water, and react at 25°C 12 hours. Add 10 units of restriction endonucleases NdeI (Cat. No.: R0111S, NEB), MseI (Cat. No.: R0525S, NEB) and BfaI (Cat. No.: R0568S, NEB) to the above reaction system, and react at 37°C for 12 hours . Heat at 80°C for 20 minutes to inactivate the above restriction endonucleases.

Embodiment 3

[0057] Example 3 Telomere DNA Double-Strand Region Affinity Modification and Single-Strand Region Affinity Probe Labeling

[0058] 3.1 Digoxigenin modification of telomeric DNA ends

[0059] Take 2 μg of genomic DNA digestion product, add 2 μl 10×0NEB buffer 2, and add dATP (product number: 4026Q, Baoriyi Biotechnology Co., Ltd.) and 0.2 mM Digoxigenin-11-dUTP (product number: 11093070910, Roche), and 10 units of Klenow Fragment (3′-5′exo-) polymerase (product number: M0212S, NEB), the volume was made up to 20 μl with deionized water. The reaction mixture was placed at 37°C for 12 hours to allow the sample to fully react.

[0060] 3.2 Biotin modification of telomeric DNA ends

[0061] 10 μl (2.4 μg) of the product from the previous step was mixed with 2.4 μl (2.4 pmol) of a biotin-modified probe (Biotin-CCCTAACCCTAACCCTAA) and subjected to a gradient annealing test. The gradient annealing program was 75°C for 3 minutes; 75-25°C (decrease by 0.1°C every 8 seconds, a total of...

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Abstract

The invention relates to a monomolecular mechanics method for measuring the accurate length of human telomere. The method comprises the following steps of: firstly, extracting genome DNA of a target cell; digesting the genome by using combined enzyme digestion, and reserving telomere DNA; carrying out affinity labeling on the two ends of telomere DNA by adopting digoxin and biotin respectively soas to obtain a telomere DNA structure capable of being used for Monomolecular mechanical measurement; secondly, fixing the telomere structure between streptavidin-coated magnetic beads and the glass surface paved with the anti-digoxin antibody, and carrying out a DNA mechanical tensile experiment on a monomolecular instrument; and finally, using a worm model to perform length conversion between nanometer and basic group pairs on the actually measured telomere length, so as to realize accurate measurement of the telomere length. The invention belongs to the field of precise instrument measurement and analysis and cell molecular biology, and develops a new method for directly measuring the accurate length of telomere for cell research, health screening and clinical drug therapeutic effect evaluation.

Description

technical field [0001] The invention belongs to the fields of precision instrument measurement analysis and cell molecular biology, and in particular relates to a method for measuring the precise length of human telomeres using single-molecule force spectrum technology. Background technique [0002] Telomeres are the lifespan clock in humans, and their length determines the fate of cells. Telomeres are located at the ends of chromosomes in eukaryotic cells and play an important role in maintaining the integrity of chromosomes and controlling the cell division cycle. The DNA sequence of the telomere is a highly repetitive sequence of 5'-TTAGGG-3'. As the number of cell divisions increases, telomere length shortens due to replication loss or DNA damage repair. When the telomere is shorter than the critical length, the apoptosis mechanism will be activated, resulting in cell apoptosis. Therefore, telomeres are also known as the biological clock of cell fate. Measuring the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6804G16B5/00G16B30/00
CPCC12Q1/6804G16B5/00G16B30/00C12Q2563/131C12Q2563/143C12Q2563/149C12Q2521/301C12Q2521/101
Inventor 于仲波李旭王美杰郑薇黄威王泽瑜
Owner NANKAI UNIV
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