Rapid method for measuring length of chromosome telomere by flow cytometry

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

Active Publication Date: 2017-12-19
沃森克里克(北京)生物科技有限公司
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  • Summary
  • 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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  • Rapid method for measuring length of chromosome telomere by flow cytometry
  • Rapid method for measuring length of chromosome telomere by flow cytometry
  • Rapid method for measuring length of chromosome telomere by flow cytometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] A kind of rapid method of flow cytometry measuring chromosomal telomere length, comprises the following steps:

[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 ...

Embodiment 2

[0067] A rapid method for determining the 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 rapid 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 invention provides a rapid method for measuring the length of chromosome telomere by a flow cytometry. Operation of performing lucifugal ice bath is increased before the conventional denatured hybridization step, a telomere probe has sufficient time to enter a cell and can be hybridized with telomere DNA rapidly and effectively after the DNA is deformed, so that the hybridization efficiency is improved and the hybridization success rate is increased; 1 percent paraformaldehyde serves as a fixing liquid, so the fixing effect is better and more stable, and the use is facilitated; 70 percent deionized formaldehyde is added into a hybridization buffer solution, and the stability of the cell structure and the DNA can be protected when probe hybridization is conducted for a long time, so that the hybridization success rate is increased. Through the improvement, compared with the traditional method for measuring the length of the chromosome telomere, the method is quicker and more accurate, 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 rapid method for measuring the length of chromosomal telomeres by flow cytometry. 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 div...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N15/10G01N21/64
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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