Telomere length detection

Telomere length detection is a special structure at the end of linear genomic DNA in eukaryotes. It is a complex formed by a DNA sequence and a protein, which prevents fusion between normal chromosome ends, ensures the integrity of each chromosome, controls cell growth and division cycle, and is closely related to cell apoptosis, cell transformation, and immortalization.

　　Telomere length detectionTelomere is a special structure at the end of the linear genome DNA in eukaryotes. It is a complex formed by a DNA sequence and a protein, which prevents fusion between normal chromosome ends, ensures the integrity of each chromosome, controls cell growth and division cycle, and is closely related to cell apoptosis, cell transformation, and immortalization. The human telomere repeat sequence DNA is composed of repeated TTAGGG sequences (see figure on the right).

During the division process of normal human cells, DNA replication cannot replicate the last small segment of its sequence, resulting in a slight shortening of DNA after each cell division, known as "chromosome end retraction". Due to the protection of telomere sequences at the end of DNA, DNA replication only loses a portion of the telomere sequence, without endangering the normal DNA sequence. When cells replicate multiple times and telomeres shorten to a certain extent, the normal DNA sequence becomes unstable, and at this point, cells begin to age and die. Research has shown that under normal circumstances, human cells will necrotize due to the production of toxins after an average of about 50 rounds of division and replication, which is known as the "Hayflick limit".

This product kit uses dual color fluorescent qPCR (SYRBgreen+VIC) to detect the relative length of telomeres, and the detection objects are blood, oral swabs, or cellular DNA. This reagent kit has the following characteristics.

1. Stability: Closed pipe operation throughout the entire process, without cross contamination.

2. Reliable: Telomere and internal reference single tube detection reduces inter well interference.

The reference gene has multiple copies and the difference in telomere size is reduced.

3. Convenience: Easy to operate, no need for electrophoresis steps.

4. Quantification: △ CT value, which can quantitatively detect the relative length of telomeres.

　　Telomere length detectionIt is a relatively simple detection that does not require a large amount of starting DNA (about 50 ng). By measuring the telomere signal (T) and the reference single copy gene signal (S), the T/S ratio is calculated, which is proportional to the average TL (Telomere Length) and can be used to determine the relative TL. Due to the nature of this technology, it can be applied to high-throughput detection and is therefore widely used in large-scale population studies. However, since Q-PCR only provides relative quantification, data is typically not presented in absolute telomere length in kb form unless compared to a reference cell line with an average TL determined by another method. There are studies showing that the coefficient of variation of the measurement results of this method may be higher than 10%. Moreover, due to the use of different single copy genes, there may be significant differences in results between different laboratories. In addition, Q-PCR does not provide information about the shortest telomeres. Finally, Q-PCR used for TL measurement may not be suitable for cancer research, as the internal reference single copy genes may be replicated or lost due to aneuploidy. Therefore, the applicability of Q-PCR is limited to samples with normal diploid and stable karyotype.

Telomeres are non coding tandem repeat (TTAGGG) n sequences located at the ends of linear chromosomes in all vertebrates. The 3 'end single stranded dangling of telomeres invades double stranded DNA, forming a T loop, which also leads to strand displacement and the formation of a single stranded telomere D-loop. The T-loop is a special structure of telomeres that directly or indirectly binds to the telomere protective protein Shelterin complex, protecting the chromosome ends from being recognized as DNA double strand breaks.

Telomeres are a special structure composed of repetitive DNA sequences and specific telomere binding proteins at the end of eukaryotic cell chromosomes. Among them, the telomeres of vertebrates are composed of short double stranded repeat sequences rich in g, called ttaggg, connected in series. Telomeres can prevent degradation, fusion, and rearrangement of chromosome ends, thereby maintaining chromosome independence, integrity, and stability. Although telomeres do not have coding functions, they are called the "clock of life" by scientists because their length and stability control the lifespan of cells and are closely related to cancer and aging.

In normal human somatic cells, the length of telomeres gradually shortens with cell division. Each time the cell divides, the length of telomeres shortens by a segment, losing 20-30 nucleotides. When telomeres shorten to a certain extent, it induces the loss of nuclear protein structure, triggering replicative aging, resulting in slow cell proliferation, growth arrest, reduced stemness, and loss of differentiation ability. At the same time, the loss of certain telomere specific binding proteins on shortened telomeres can also lead to cells misidentifying telomere ends as DNA break sites, thereby initiating DNA repair function, resulting in end fusion between short telomere chromosomes. Chromosome fusion can cause abnormal cell division, cell cycle arrest, and ultimately trigger apoptosis induced by p53 protein.

In addition, mutations in genes such as p53 can lead to defects in cell cycle checkpoint, causing cells to undergo replicative aging, continue to divide, and eventually enter a crisis period. During this period, a very small number of cells express telomerase, activate telomerase activity, repair and maintain telomere length, and make cells immortal into cancer cells. Telomere over shortening and telomerase activity were found in 90% of cancer cells, such as rectal cancer, breast cancer, lung cancer, prostate cancer, etc. Diseases caused by telomere abnormalities also include leukemia, aplastic anemia, abnormal bone marrow proliferation, and congenital keratinization. Telomeres play an extremely important role in the process of human aging and disease occurrence. The length changes of telomere repeat sequences determine the fate of cells, thereforeTelomere length detectionIt is an important experimental method in telomere biology research.

In addition,Telomere length detectionIt also has important diagnostic value in aging diseases and tumors. Due to species differences, the telomere length of human chromosomes (double stranded region length generally ranging from 0.5 to 20kb) is much smaller than that of most laboratory model organisms; And the degree of telomere shortening varies among different individuals and cells, as well as among different chromosomes within the cell; Telomeres with a length of less than or equal to 3kb are defined as short telomeres, and the shortest telomere, rather than the average telomere length, plays an important role in telomere dysfunction, inducing DNA damage, and limiting cell survival. Therefore, there is an urgent need for a method that can accurately, simply, quickly, and high-throughput detect human telomere length and short telomere ratio at the level of a single chromosome.

During the development of telomere related genetic diseases, having shorter telomeres can lead to early onset of the disease. With the continuous deepening of research, people are increasingly aware that lifestyle factors such as obesity, smoking, lack of exercise, and chronic stress can affect the telomere length in circulating peripheral blood white blood cells. In addition, telomere shortening, which leads to telomere dysfunction, is associated with many age-related diseases, such as infertility, arthritis, diabetes, cancer, cardiovascular and neurodegenerative diseases. Therefore, stable and reproducible methods for measuring telomere length that can predict the occurrence of these diseases are crucial.

However, almost all published large-scale population studies on telomere related diseases are essentially correlation studies, providing only information on average or relative telomere length. There is ample evidence to suggest that the shortest telomeres trigger the DNA damage response, leading to replicative aging in mammals. The length of the shortest telomere is a key biomarker that determines cell fate and the onset of aging, but methods for detecting short telomeres are time-consuming and laborious, and cannot meet high-throughput requirements. Therefore, existing methods for measuring telomere length have their own advantages and disadvantages.

Abnormal changes in telomere length may occur in the early stages of diseases, and telomere length may become an early screening indicator, even an auxiliary diagnostic indicator or prognostic evaluation indicator for certain diseases decades later. Simple and feasible high-throughput detection methods can be used for large-scale epidemiological studies to identify telomere related diseases, providing more accurate detection methods for different telomere length distributions that can be used to search for possible mechanisms between telomere length and related diseases; Of course, a high-throughput, accurate, and multi-faceted telomere detection method should be our research goal.