PCR method for detecting absolute telomere length

A technology of telomere length and telomere, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem that the absolute length of telomere cannot be obtained, and achieve low detection cost, accurate detection results, and high throughput Effect

Inactive Publication Date: 2019-06-28
山东荆卫生物科技有限公司
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Problems solved by technology

[0005] Purpose of the invention: In order to solve the problem that the Q-PCR method detects the telomere length, only the relative length of the telomere can be obtained, b

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  • PCR method for detecting absolute telomere length
  • PCR method for detecting absolute telomere length
  • PCR method for detecting absolute telomere length

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[0040] Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

[0041] The absolute telomere length of human lymphocyte DNA detected by the present invention is specifically described below:

[0042] (1) Separation of human lymphocytes: collect 2ml of fresh anticoagulated peripheral blood, and use GE Ficoll-Paque PLUS lymphocyte separation medium to separate lymphocytes;

[0043] (2) Genomic DNA extraction: Take isolated lymphocytes, use TIANGEN blood / cell / tissue genomic DNA extraction kit...

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Abstract

The invention provides a PCR method for detecting absolute telomere length, and belongs to the field of molecular biological techniques and medical detection. According to the method, the absolute length of the telomere of the human can be conveniently and accurately detected. A gene synthesis method is adopted for synthesizing oligonucleotide sequences of the telomere and a single-copy gene 36B4respectively, the sequences are connected to a plasmid vector to construct a standard product, escherichia coli is utilized for in-vitro amplification of the plasmid standard product, the plasmid DNAis extracted, the concentration is measured, and the copy number and the total length of the telomere are calculated. The initial mass of the standard product is determined, gradient dilution is conducted, a fluorescence quantitative PCR method (Q-PCR) is adopted for obtaining standard curves of the telomere and the 36B4 separately, through the standard curves, the total length of the telomere andthe copy number of the 36B4 of the to-be-tested sample DNA are subjected to absolute quantification, and thus the average absolute telomere length of the to-be-tested sample DNA is calculated. The length of the telomere is detected through the absolute telomere length PCR method, and the PCR method has the advantages of being low in cost, short in period and high in accuracy, and is suitable forhigh-throughput detection, easy to clinically apply and popularize, and high in practicability.

Description

technical field [0001] The present invention relates to the fields of molecular biotechnology and medical detection, and in particular to a method for detecting the absolute length of human telomeres. By means of fluorescent quantitative PCR method, standard curves are prepared by constructing telomeres and single-copy gene 36B4 plasmid standards to realize The detection of human absolute telomere length, the method is called absolute telomere length PCR (aTL PCR). Background technique [0002] Telomere is a special structure at the end of eukaryotic chromosome, which is composed of DNA and telomere protein. Telomere DNA is a highly conserved repeating nucleotide sequence rich in G. The telomere sequences of different species are not consistent. Human chromosomal telomeres are composed of simple repeating (TTAGGG) sequences in the 5'→3' direction, and the telomere length is 0.5-20kb. Telomeres are non-structural genes that do not have the function of coding proteins, but ha...

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Application Information

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IPC IPC(8): C12Q1/6851
Inventor 白玉杰林思妮孙海星张艳张晓娟王建辉董红宇吴东颖
Owner 山东荆卫生物科技有限公司
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