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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (8): 63-71    DOI: 10.13523/j.cb.2211008
技术与方法     
牛绝对端粒长度测定及DNA提取造成的影响*
鲍莉雯1**(),蔡勤1**,周一叶1,2,3,许淼1,3,舒娟1,3,李华1,3,李虹雨1,3,曾溢滔1,3,曾凡一1,2,3,***()
1 上海市儿童医院 上海交通大学医学院附属儿童医院 上海医学遗传研究所 上海 200040
2 上海交通大学基础医学院组织胚胎学与遗传发育学系 上海 200025
3 卫生部医学分子生物学重点实验室 上海市胚胎与生殖工程重点实验室 上海 200040
Determination of Absolute Telomere Length in Cattle and the Effect of DNA Extraction
BAO Li-wen1**(),CAI Qin1**,ZHOU Yi-ye1,2,3,XU Miao1,3,SHU Juan1,3,LI Hua1,3,LI Hong-yu1,3,ZENG Yi-tao1,3,ZENG Fanyi1,2,3,***()
1 Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200040, China
2 Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
3 Key Laboratory of Embryo Molecular Biology, Ministry of Health and Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
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摘要：

目的:端粒是真核生物染色体末端的一种高度保守的负责维持染色体稳定的特殊结构,其DNA序列长度即端粒长度,会随着年龄增长或疾病发生发展而逐渐缩短,检测端粒长度可以为评估机体衰老和健康状况提供参考,但目前缺乏测定微量牛DNA 样本绝对端粒长度的方法;通过实时荧光定量PCR(real-time quantitative PCR, qPCR)实现微量牛DNA 样本绝对端粒长度的测定并评估DNA提取方法对牛绝对端粒长度测定结果的影响,为进行端粒长度研究时选择合适的DNA提取方法和端粒长度分析方法提供参考。方法:利用标准曲线对检测样本的端粒和内参Ct值进行转换,通过qPCR测定牛端粒长度绝对值;采用膜吸附法、苯酚-氯仿法和磁珠法3种方法分别提取相同样本的DNA,分别用端粒末端限制性片段(terminal restriction fragment, TRF)分析法和qPCR法分析端粒长度,比较不同DNA提取方法对牛绝对端粒长度测定的影响。结果:(1)qPCR可以测定纳克级别DNA样本的绝对端粒长度,检测结果重复性良好,并且和“金标准”TRF测定结果的相关性良好。(2)不同方法提取的DNA用TRF分析法和qPCR法测定端粒长度时,结果有显著差异,其中磁珠法提取的DNA在用2种不同方法进行端粒长度测定时,测定结果的一致性最好。结论:qPCR法是较 TRF分析法更加灵敏的端粒长度绝对值测定方法,适合微量DNA样本的测定,且方便快捷;DNA提取方法会影响端粒长度的测定结果,在测定时应统一样本的DNA提取方法,磁珠法是相对更优的选择。
关键词： 端粒长度;  qPCR;  TRF;  DNA提取方法    
Abstract:

Objective: Telomere is a highly conserved structure at the end of eukaryotic chromosomes responsible for maintaining chromosomal stability, and the length of its DNA sequence, known as telomere length, gradually shortens with age or disease development. Telomere length can provide a reference for assessing the aging and health status of individuals. However, there is no satisfactory method for determining the absolute telomere length from a small amount of cattle samples. Real-time quantitative PCR (qPCR) was used to determine the absolute telomere length of minute DNA samples from cows and to assess the effect of the DNA extraction method on the results of absolute telomere length determination. This study aims to provide a reference for selecting suitable DNA extraction methods and telomere length analysis methods for a study on telomere length in cattle. Methods: The absolute values of telomere length were determined by qPCR. DNA was extracted from the same samples with silica membrane, phenol-chloroform, and magnetic beads, and the telomere lengths were analyzed by terminal restriction fragment (TRF) assay and qPCR, respectively, to compare their effect on cattle absolute telomere length determination. Results: (1) qPCR can be used to determine the absolute telomere length of nano-gram level cow DNA samples with good reproducibility and correlates well with the “gold standard” TRF results. (2) DNA extracted by different methods would result in significant differences in results when used for telomere length analysis. DNA extracted by the magnetic bead method showed the best agreement when telomere length was measured by TRF and qPCR methods. Conclusion: qPCR is a more sensitive, convenient, and rapid method for determining absolute telomere length than the TRF method and is suitable for determining minute DNA samples. The DNA extraction method can affect the results of telomere length determination and should be unified while the assay was performed, and the magnetic bead method was optimal.
Key words: Telomere length    qPCR    TRF    DNA extraction methods
收稿日期: 2022-11-07 出版日期: 2023-09-05
ZTFLH:  Q789  
基金资助: 国家重点研发计划(2019YFA0801402);国家自然科学基金(31871484);国家自然科学基金(82271890)
通讯作者: ***电子信箱:fzeng@vip.163.com   
作者简介: ** 并列第一作者
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鲍莉雯
蔡勤
周一叶
许淼
舒娟
李华
李虹雨
曾溢滔
曾凡一

引用本文:

鲍莉雯, 蔡勤, 周一叶, 许淼, 舒娟, 李华, 李虹雨, 曾溢滔, 曾凡一. 牛绝对端粒长度测定及DNA提取造成的影响*[J]. 中国生物工程杂志, 2023, 43(8): 63-71.

BAO Li-wen, CAI Qin, ZHOU Yi-ye, XU Miao, SHU Juan, LI Hua, LI Hong-yu, ZENG Yi-tao, ZENG Fanyi. Determination of Absolute Telomere Length in Cattle and the Effect of DNA Extraction. China Biotechnology, 2023, 43(8): 63-71.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2211008    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I8/63

图1  端粒和内参标准曲线
图2  qPCR法测定不同年龄段牛耳皮肤端粒长度结果比较
图3  qPCR测定端粒长度的重复性
序号 端粒检测方法 TRF qPCR
1 需要DNA量/μg ≥4 0.12
2 耗时/h(1) 120[18] 5
3 灵敏度(检测下限)/ kb 约2 [17] 约0.16
4 组内变异系数(2)(端粒&内参)/% NA 1.48 & 0.86
5 批间变异系数(3)(n=26(4))/% <2[18] 6.06
6 对DNA完整性的依赖程度 高 低
表1  端粒长度测定的TRF分析法和qPCR法比较
样本编号 年龄/岁 分组
1 0.77 低龄组
2 0.73 低龄组
3 1.28 低龄组
4 1.07 低龄组
5 12.00 高龄组
6 12.00 高龄组
7 11.00 高龄组
8 11.00 高龄组
9 0.04 低龄组
10 1.87 低龄组
表2  牛耳皮肤样本信息
序号 提取方法(组别) M1 M2 M3
1 初始组织样本数 10 10 10
2 平均起始组织量/(mg/份) 63.55±3.86a 63.52±3.89a 61.17±2.74a
3 平均DNA回收量/(μg/份) 34.87±17.12a 122.09±29.77b 33.34±6.44a
4 DNA质量合格样本数(1) 10 9(2) 8(2)
5 DNA完整性评分=1分的样本数(1) 0 6 8
表3  三种不同DNA提取方法比较
图4  不同方法提取DNA的完整性鉴定
图5  TRF分析法和qPCR法测定牛端粒长度结果
图6  TRF分析法和qPCR法测定端粒长度结果相关性
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