PCR方法检测端粒长度

发布时间：2018-02-26 20:33

本文关键词： 端粒年龄实时定量PCR　出处：《昆明医学院》2006年硕士论文　论文类型：学位论文

【摘要】：目的端粒在细胞正常生理功能运转过程中起重要作用，同一组织端粒的长度变化与年龄呈负相关，即随着年龄的增加，端粒长度均表现为逐渐变短的趋势，这种变化趋势表现为组织特异性，即同年龄不同组织细胞端粒的长度不同(在生殖细胞中，由于端粒酶的作用，端粒长度保持不变)，而且同一组织细胞端粒长度随年龄缩短不均衡，，并且发现这种不均衡大体分为三个阶段，婴幼儿时期端粒缩短趋势最快，青少年时期减缓，至壮年以后速度有所回升。我们采取不同年龄阶段同一组织(外周血)样本排除组织差异后，利用实时定量PCR的方法通过T/S的比值检测相对端粒长度，如果检测的样本量足够大，就有可能建立起端粒长度与年龄的相关曲线以及方程。方法 278例外周血样本用改进后的酚、氯仿、异戊醇法提取DNA，采用PCR方法，扩增不同年龄阶段样本的端粒片断，测序检测确定为目的片段，再用实时定量PCR检测端粒相对长度。结果普通PCR扩增出长度大约为200～600bp之间的多条端粒片断，测序结果显示为目的片段，实时定量PCR可以扩增端粒片断，利用实时定量PCR检测端粒相对长度的方法可行。结论 PCR的方法可以扩增端粒片断，与经典的Southern杂交方法相比具有：简便、快捷、操作容易、实验室费用消耗较低等特点。并且通过本次试验对用实时定量PCR的方法检测端粒相对长度，做了部分尝试，认为该方法可行，为后续试验做好准备。
[Abstract]:Objective telomere plays an important role in the normal physiological function of cells. The change of telomere length in the same tissue is negatively correlated with age. This trend shows tissue specificity, in which telomeres vary in length at the same age (in germ cells, due to the role of telomerase). Telomere length remained unchanged, and the length of telomere in the same tissue cells decreased unevenly with age. It was found that the imbalance was divided into three stages: telomere shortening was the fastest in infancy and slowed down in adolescence. After reaching adulthood, we used the same tissue (peripheral blood) samples at different ages to exclude the difference of tissue, and measured the relative telomere length by the ratio of T / S by real-time quantitative PCR. If the sample size is large enough, it is possible to establish the correlation curve and equation between telomere length and age. Methods two hundred and eighty-eight peripheral blood samples were extracted by modified phenol, chloroform and isoamyl alcohol method. The telomere fragments were amplified by PCR method and sequenced. The relative length of telomere was detected by real-time quantitative PCR. Results A number of telomere fragments with a length of about 200 ~ 600bp were amplified by ordinary PCR. The results of sequencing showed that telomere fragments could be amplified by real-time quantitative PCR. The method of detecting the relative length of telomere by real-time quantitative PCR was feasible. Conclusion the method of PCR can amplify the telomere fragment. Compared with the classical Southern hybridization method, it is simple, fast and easy to operate. The cost of laboratory is low, and some attempts have been made to detect the relative length of telomere by the method of real-time quantitative PCR, and it is considered that this method is feasible and ready for further test.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：D919

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