Methods for measuring telomere length

Abstract

The present disclosure relates to a method for detecting telomere length, the method comprising the following steps: S1. Pretreating the cell sample to be tested to obtain the M phase chromosome suspension of the cell sample to be tested; S2. Centrifuging the chromosome suspension and removing the supernatant to obtain the first precipitate containing chromosomes in the M phase, mixing the first precipitate with a hybridization solution containing a telomere-specific peptide nucleic acid fluorescent probe, performing denaturation and hybridization to obtain a suspension containing hybrid chromosomes; S3. Washing, centrifuging, and removing the supernatant of the hybrid chromosome suspension to obtain a second precipitate containing the hybrid chromosome, suspending the second precipitate with a suspension medium to obtain a sample loading suspension; S4. Using ultra-high sensitivity flow Cytometry detects the fluorescent signals of the chromosomes in the detection loading suspension one by one at the single particle level. The method can realize rapid, accurate and quantitative detection of human telomere length and short telomere ratio at the single-chromosome level.

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...

AI Technical Summary

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

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