| 摘要 |
The invention is intended for identifying changes in nucleotide terminals of the DNA of the telomeric regions of chromosomes occurring during the course of the cell cycle. The method is based on a novel approach without the use of ligation. The method comprises the steps of first extracting genome DNA, isolating a fraction of single-stranded G overhangs of telomeric DNA and subsequently amplifying the minus chain of said G overhangs with duplex-specific analysis. The amplification steps comprise modification of the 3' ends of telomeric G overhangs with the aid of a terminal deoxynucleotidyl transferase and are performed using a set of primers in sequences SEQ ID NO:1-5. The duplex-specific analysis is based on hybridization of the minus chains of the overhangs with a set of specific fluorescent probes (SEQ ID NO:6-11), which correspond to six possible variants of the nucleotide terminals of the G chain of human telomeric DNA. The degree of buildup of the probes in the presence of a duplex-specific nuclease enzyme is used as the criterion for the presence of a corresponding variant of a nucleotide terminal of DNA and as a measure of the quantitative content thereof. The profiles obtained for the percentage content of terminal triplets of the G chain of telomeric DNA are used as proliferative markers indicating the degree of activation of cell division and determining the duration of the cell cycle. The invention makes it possible to increase sensitivity and accuracy in the quantitive analysis of terminal nucleotides in comparison with procedures based on ligation. The method makes it possible to produce diagnostic results in immunology, oncology, organ transplantation, cosmetology, dermatology, gerontology, cell bioengineering and other fields interested in the assessment of the proliferative status of cells and procedures for the targeted regulation thereof. |
