The Method of Measuring the Absolute Length of Chromosome Telomeres by Single-color Multiplex Quantitative PCR

A chromosome and telomere technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of measurement accuracy of a large number of DNA samples, absolute length of chromosomal telomeres, time-consuming and labor-intensive problems, etc., to achieve easy operation, strong consistent effect

Inactive Publication Date: 2011-12-28
杨楠 +1
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Problems solved by technology

[0004] The purpose of the present invention is to provide a method for measuring the absolute length of chromosomal telomeres by a single-color multiplex quantitative PCR method, which solves the p

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  • The Method of Measuring the Absolute Length of Chromosome Telomeres by Single-color Multiplex Quantitative PCR
  • The Method of Measuring the Absolute Length of Chromosome Telomeres by Single-color Multiplex Quantitative PCR
  • The Method of Measuring the Absolute Length of Chromosome Telomeres by Single-color Multiplex Quantitative PCR

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[0073] Example

[0074] 1. Design and synthesis of primers

[0075] The primer design was synthesized by the phosphoramidite triester method according to the literature.

[0076] 2. Drawing the standard curve

[0077] Replace genomic DNA with serially diluted telomeres and single-copy gene standard sequences in a standard sample reaction well. To make up for the diluted standard sequence for less than 20ng, add pBR322 plasmid DNA to the standard reaction wells to make the standard sample wells There are 20ng of DNA. The standard sample hole is used to make a standard curve. The following are described separately:

[0078] Telomere standard curve:

[0079] Synthesize 84-mer (TTAGGG) with a known number of molecules 14 Perform serial dilutions, and the number of replicates for each standard is calculated as follows:

[0080] The length of the standard sequence is 84bp (TTAGGG is repeated 14 times), and the molecular weight (MW) is 26667.2.

[0081] The mass of a single standard sequence m...

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Abstract

The invention discloses a method for measuring the absolute length of chromosomal telomeres by single-color multiplex quantitative PCR, which is characterized in that the method comprises the following steps: (1) designing and screening primers for chromosomal telomeres and primers for amplifying internal reference genes Single-copy gene primers require that the difference between the Tm values ​​of the telomere gene primers and the single-copy gene primers be in the range of 10-20°C; (2) Take the telomere sequence of SEQNo: 1 and the single-copy gene of SEQNo: 2 as Standard sequence, draw a standard curve by the external standard method; (3) extract the template DNA, add the template DNA to the PCR reaction system with the primers in step (1) to perform PCR amplification of the target gene in the template DNA; (4) use the standard The curve is the control. The absolute length of telomere was determined by quantitative PCR method. This method eliminates the sampling error, makes the results more accurate, and breaks through the limitation that ordinary quantitative PCR can only measure the relative length of telomeres.

Description

technical field [0001] The invention belongs to the technical field of gene measurement, and in particular relates to a method for measuring the absolute length of chromosomal telomeres by a single-color multiple quantitative PCR method. Background technique [0002] Telomeres (tolemere) are DNA repeats at the ends of chromosomes, which are tandem repeats of (TTAGGG) in human chromosomes. Telomeres are specialized structures at the ends of chromosomes that, in normal human cells, gradually shorten as cells divide. The role of telomeres is to maintain the integrity of chromosomes. Once the cell divides, because the direction of DNA replication must be from 5' to 3', the telomere shortens a little each time the DNA replicates. Once telomeres are depleted, chromosomes are prone to mutations that lead to arteriosclerosis and certain cancers. In recent years, a variety of telomere length measurement methods have been reported, including Southern blot (SB), hybridization protec...

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 杨楠艾洪新杨林
Owner 杨楠
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