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Method for detecting telomere length

A technology of telomere length and detection end, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high technical requirements, large error of tumor cells, and inability to obtain short telomere ratio, and achieve high sensitivity, Effects without background distractions

Active Publication Date: 2018-09-04
XIAMEN UNIV
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Problems solved by technology

This method is only applicable to diploid cells with complete nuclei and mitotic interphase cells and samples with known and stable karyotypes. For tumor cells with highly variable karyotypes, the error is extremely large; PNA probes will non-specifically bind to the cytoplasm of cells Therefore, it is better to use complete nuclei instead of cells; in addition, this method can only evaluate the average telomere length in a single cell, and cannot provide information on short telomeres, and requires high technical requirements and time-consuming Difficult to operate
[0011] The limitations of the above method are: 1) limited by the principle of the method, only the average length of telomeres can be detected, and the ratio of short telomeres cannot be obtained, such as TRF, qPCR and Flow-FISH; 2) limited by the detection probe binding efficiency and / or detection technique sensitivity, unable to detect shorter telomeres, such as TRF, qPCR and Flow-FISH; 3) limited by photobleaching and / or lack of good reference standards, unable to accurately measure telomere length, such as Q- FISH and Flow-FISH; 4) are specific, but not broad-spectrum, and can only detect the telomere length of specific chromosomes, such as STELA; 5) can detect abnormally short telomeres of a single chromosome, but are not suitable for epidemic Disease research (not Qualcomm), such as Q-FISH
Therefore, there is currently no single method that can accurately, simply, quickly, and high-throughput measure telomere length and its distribution and short telomere ratio

Method used

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  • Method for detecting telomere length
  • Method for detecting telomere length
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Examples

Experimental program
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Embodiment 1

[0108] This example is used to illustrate the use of telomere-specific peptide nucleic acid fluorescent probe (positive probe: 5'-Alexa Fluor488-(CCCTAA) 3 -3') and peptide nucleic acid fluorescent probes with 3 base changes (negative probe: 5'-Alexa Fluor 488-(GCCTAA) 3 -3') Verify that there is no non-specific adsorption and mismatch in the probe, which will not affect the detection results. The operation flowchart reference of this embodiment figure 1 .

[0109] The laboratory cultured cell strain used in this example is HeLa, and its culture method is: place the cells in a petri dish containing 6 mL of DMEM medium for 24 hours of adherent culture, and then add autumn 1 mg / L to the petri dish. The cells were stimulated with narcissine for 8 hours.

[0110] Centrifuge and enrich 6 mL of the above-mentioned medium containing HeLa suspension cells in the M phase at a centrifugation speed of 350 rcf and a centrifugation time of 5 minutes. 4 , 5mmol / L HEPES and 3mmol / L β-mer...

Embodiment 2

[0116] This example is used to illustrate the detection of the telomere length and short telomere ratio of 5 kinds of laboratory cultured cell strains by the method provided by the present disclosure, and the average length of telomere of 5 kinds of laboratory cell strains obtained by the method provided by the present disclosure Comparison of assay results with "gold standard" telomeric end-restriction fragment analysis.

[0117] The five selected laboratory cell lines are HeLa, A549, HEK293T, SMMC-7721 and HepG2. The HEK293T is an immortal cell growing semi-adherently, and the other four are tumor cells growing adherently.

[0118] In this example, the method of laboratory preparation of chromosome samples and probe hybridization is consistent with that of Example 1. Five laboratory cell lines were cultivated using the same cell culture method as in Example 1, wherein the HeLa, HEK293T and HepG2 used DMEM medium, and the A549 and SMMC-7721 used RPMI-1640 medium.

[0119] Th...

Embodiment 3

[0125] This example is used to illustrate the detection of the telomere length and short telomere ratio of normal human peripheral blood lymphocytes by the method provided in the present disclosure.

[0126] The normal human peripheral blood sample used in this embodiment comes from a healthy adult, male, aged 25 years. The peripheral blood of normal people is collected using a vacuum blood collection tube containing heparin anticoagulant, and blood is collected from the cubital fossa vein.

[0127] The cells used in this embodiment are the lymphocytes in the peripheral blood mononuclear cells, and the peripheral blood mononuclear cells are separated using Ficoll-Paque PREMIUM (density: 1.077g / mL) separation medium (purchased from GE Company), and the peripheral blood single The extraction method of nuclear cells was carried out according to the instruction manual of Ficoll-Paque PREMIUM (density: 1.077g / mL) product of GE Company.

[0128] In this example, the method for cult...

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Abstract

The invention relates to a method for detecting telomere length. The method comprises the following steps: S1, pretreating a cell sample to be detected to obtain a M-phase chromosome suspension of thecell sample to be detected; S2, centrifuging the chromosome suspension, removing a supernatant to obtain a first precipitate containing M-phase chromosomes, mixing the first precipitate with a hybridization solution containing a telomere-specific peptide nucleic acid fluorescent probe, denaturing and hybridizing to obtain a suspension containing the hybrid chromosomes; S3, washing and centrifuging the hybrid chromosome suspension, removing a supernatant to obtain a second precipitate containing the hybrid chromosomes, suspending the second precipitate by using a suspending medium to obtain adetection sample suspension; S4, detecting fluorescent signals of the chromosomes in the detection sample suspension one by one in a single-particle level by an ultrahigh-sensitivity flow cytometry. By the method, fast, accurate and quantitative detection of the human telomere length and the short telomere ratio in a single-chromosome level can be achieved.

Description

technical field [0001] The present disclosure relates to a method of detecting telomere length. Background technique [0002] The 2009 Nobel Prize in Physiology or Medicine was awarded to three scholars, Elizabeth Blackburn, Carol Grad and Jack Szostak, in recognition of their discovery of the special structure of telomeres, the chromosome protection function of telomeres and Telomerase and its mechanism of action. [0003] Telomere is a special structure composed of repetitive DNA sequence and special telomere binding protein at the end of eukaryotic cell chromosome. The telomere of vertebrate is composed of G-rich short double-stranded repeat sequence TTAGGG in series. Telomeres can prevent the degradation, fusion and rearrangement of chromosome ends, thereby maintaining the independence, integrity and stability of chromosomes. Although telomeres do not have coding functions, they are called "the clock of life" by scientists, because the length and stability of telomeres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6816
CPCC12Q1/6816C12Q2563/107C12Q2525/107C12Q2565/113
Inventor 颜晓梅周颖星高恺旻
Owner XIAMEN UNIV
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