Telomere length detection method based on fluorescent quantitative PCR

A technique for quantifying telomere length and fluorescence, which is applied in the field of molecular biology, can solve problems such as inconvenient detection methods for telomere length, unsuitable for clinical promotion, and inaccurate telomere length, so as to prevent aging and cancer with small conversion errors , the effect of high fitting degree

Inactive Publication Date: 2013-06-26
北京海斯凯生物科技有限公司
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Problems solved by technology

[0009] Recently, some scholars (O'Callaghan NJ, Biol Proced Online, 2011) used an oligonucleotide fragment of known fragment size as a length calibration standard to quantify the absolute length of other genomic telomeres, but this method also has some limitations. Disadvantages: 1) Because it is an oligonucleotide fragment with a relatively simple structure, the efficiency of PCR may be different when amplifying oligonucleotides and genomic DNA. Therefore, oligonucleotides are used as calibration markers,

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  • Telomere length detection method based on fluorescent quantitative PCR
  • Telomere length detection method based on fluorescent quantitative PCR
  • Telomere length detection method based on fluorescent quantitative PCR

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[0038] The specific detection process of the telomere length detection method based on the fluorescent quantitative PCR of the present invention is divided into the following steps.

[0039] (1) Extract genomic DNA

[0040] The extracted genome can come from specimens from blood, buccal swabs, sperm, eggs, tissues, etc. The extracted genomic DNA should be of high purity and can be extracted using silica gel membrane spin columns or magnetic bead separation methods.

[0041] Taking the blood genomic DNA extraction kit produced by Beijing Bohai Tongda Biotechnology Co., Ltd. as an example, the specific steps for extracting genomic DNA are as follows:

[0042] a) Take 200μl of blood sample to which anticoagulant has been added;

[0043] b) Add 20μl ProteinaseK solution and mix well;

[0044] c) Add 200μl of binding buffer PBB1, fully invert and mix well, and place it at 56°C for 10 minutes. During this period, invert and mix several times, the solution should be clear;

[0045] d) Add 200μl ...

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Abstract

The invention provides a telomere length detection method based on fluorescent quantitative PCR. The method comprises the following steps of: extracting genome DNA (deoxyribonucleic acid); carrying out fluorescent quantitative PCR detection, detecting telomere repetitive sequence by using primers SEQ ID NO.1 and SEQ ID NO.2, and detecting 36B4 gene sequence of human acidic ribosomal phosphor-protein PO by using primers SEQ ID NO.3 and SEQ ID NO.4; calculating a relative telomere length T/S; and further calculating the absolute length of the telomere according to the derived formula. In the formula, Eva Green is used as dye, the telomere primers and internal control primers are used for detecting the telomere length by using the fluorescent quantitative PCR method, and the conversion relation between telomere TRF length and the T/S ratio is derived based on the equation of linear regression of large sample analysis, and therefore the telomere length detection method which is suitable for clinic is provided.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for detecting telomere length based on fluorescent quantitative PCR. Background technique [0002] Telomere is a specific structure at the end of chromosomes, which has the functions of preventing chromosomes from recombining and fusing, and protecting chromosomes from being degraded by nucleases. Human chromosome telomeres are composed of thousands of short repeat sequences of TTAGGG, and the total length is generally 5-20kb. However, due to DNA replication, every time a cell divides, the telomeres are shortened by about 150bp. When telomeres shorten to a certain extent, cells cannot continue to divide and enter death. [0003] In order to avoid this death caused by DNA replication, in some specific human cells such as hematopoietic stem cells and germ cells, a component called telomerase (telomerase) is used to replicate the telomere sequence to keep it constant leng...

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 辛忠涛牛宇辰崔雷房岳张英民
Owner 北京海斯凯生物科技有限公司
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