Method of Measuring Chromosome Telomere Length by Flow Cytometry

A technology of flow cytometry and telomere length, applied in the field of molecular biology, can solve the problems of unsuitable long-term large-scale research work, low accuracy, complicated operation, etc., to achieve good fixation effect, improve efficiency and success rate , easy to use effect

Active Publication Date: 2021-07-30
沃森克里克(北京)生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods all have defects such as time-consuming, cumbersome operation, and low precision, and are not suitable for long-term and large-scale research work.

Method used

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  • Method of Measuring Chromosome Telomere Length by Flow Cytometry
  • Method of Measuring Chromosome Telomere Length by Flow Cytometry
  • Method of Measuring Chromosome Telomere Length by Flow Cytometry

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Embodiment 1

[0060] A method for measuring the length of chromosomal telomeres by flow cytometry, comprising the steps of:

[0061] S1: Cell sample pretreatment: collect the single cell suspension of the cells to be tested, fix in an ice bath after washing, and elute the fixative after the fixation is completed to obtain the cell samples to be tested;

[0062] S2: Telomere hybridization: Add telomere hybridization solution containing 0.3 mg / ml telomere probe to the cell sample to be tested, and conduct ice bath under dark conditions, take it out, perform denaturation in high-temperature water bath, and keep in dark 1. Hybridization is carried out under room temperature conditions to obtain a hybridization sample to be tested, and the base sequence of the telomere probe is as follows:

[0063] Tel-PNA: FITC-Lys-Lys-CCCTAACCCTAACCCTAA;

[0064] S3: Washing for later use: adding an eluent to the hybridization sample to be tested for elution, a total of three elutions, discarding the eluent, ...

Embodiment 2

[0067] A method for measuring telomere length of chromosomes by flow cytometry, comprising the operation steps described in Example 1, wherein the fixative used is 1% paraformaldehyde.

Embodiment 3

[0069] A method for measuring telomere length of chromosomes by flow cytometry, comprising the operation steps described in Example 2, wherein the eluent used for elution is phosphate buffer containing 5% calf serum and 0.1% saponin.

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Abstract

The present invention provides a fast method for determining the length of chromosome telomere by flow cytometry, adding an operation of avoiding light and ice bath before the conventional denaturation hybridization step, so that the telomere probe has enough time to enter the inside of the cell, and After the DNA is deformed, it can quickly and effectively hybridize with the telomeric DNA, improving the efficiency and success rate of hybridization; using 1% paraformaldehyde as the fixative, the fixation effect is better, more stable, and more convenient to use; in the hybridization buffer Adding 70% deionized formaldehyde in the probe can protect the stability of cell structure and DNA during long-term probe hybridization, so as to improve the success rate of hybridization. Through the above improvements, the method is faster and more accurate than the traditional method for measuring the length of chromosomal telomeres, and is suitable for long-term and large-scale research work.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a method for measuring chromosome telomere length by flow cytometer. Background technique [0002] Telomere is a short multi-repeated non-transcribed sequence (TTAGGG) and some binding proteins to form a special structure. In addition to providing a buffer for non-transcribed DNA, it can also protect the ends of chromosomes from fusion and degradation. It plays a role in chromosome positioning, replication, It plays an important role in protecting and controlling cell growth and lifespan, and is closely related to cell apoptosis, cell transformation and immortalization. When a cell divides, the telomeres of each chromosome get progressively shorter. A part of the gene that constitutes the telomere is about 50-200 nucleotides, which cannot be completely replicated (lost) due to multiple cell divisions, so that the cell stops its function and no longer divides. Severely...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6816G01N15/10G01N21/64
CPCC12Q1/6816G01N15/10G01N21/6486G01N2015/1006C12Q2565/626C12Q2525/151C12Q2563/107
Inventor 周菊华
Owner 沃森克里克(北京)生物科技有限公司
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