Non-radioactive TRF assay modifications to improve telomeric DNA detection efficiency in plants.
Non-radioactive TRF assay modifications to improve telomeric DNA detection efficiency in plants.
复制标题
非放射性 TRF 测定修改可提高植物端粒 DNA 检测效率。
DOI：
10.1007/s12668-016-0223-z
复制
发表时间：
2016
期刊：
影响因子：
3
通讯作者：
Shakirov,EugeneV
中科院分区：
文献类型：
--
作者：
Nigmatullina,LiliiaR;Sharipova,MargaritaR;Shakirov,EugeneV
端粒DNA的长度通常被认为是衰老和一般健康状况的细胞生物标志物。已经开发了几种端粒长度测量测定法，其中最常见的是端粒限制性片段（TRF）分析，其通常涉及使用放射性标记的寡核苷酸探针。虽然这种方法非常有效，但可能会造成严重的健康问题，并产生放射性废物。地高辛（DIG）替代放射性探针已被开发和成功地用于许多测定。在这里，我们优化了DIG协议来测量端粒长度的模式植物拟南芥和目前的证据表明，这种方法可以成功地用于有效和准确地测量植物端粒长度。具体地，42 °C而不是典型的55 °C的杂交温度似乎产生更强的信号。此外，在标记寡核苷酸的5′-末端而不是3′-末端掺入DIG大大增强了信号。我们的结论是，非放射性TRF检测可以作为有效的放射性方法在检测和测量端粒长度的植物，使这种测定适合于医疗和研究实验室无法利用放射性，由于危险废物处理和安全问题。
The length of telomeric DNA is often considered a cellular biomarker of aging and general health status. Several telomere length measuring assays have been developed, of which the most common is the telomere restriction fragment (TRF) analysis, which typically involves the use of radioactively labeled oligonucleotide probes. While highly effective, this method potentially poses substantial health concerns and generates radioactive waste. Digoxigenin (DIG) alternatives to radioactive probes have been developed and used successfully in a number of assays. Here, we optimize the DIG protocol to measure telomere length in the model plantArabidopsis thalianaand present evidence that this approach can be used successfully to efficiently and accurately measure telomere length in plants. Specifically, hybridization temperature of 42 °C instead of the typical 55 °C appears to generate stronger signals. In addition, DIG incorporation at 5′-end instead of 3′-end of the labeled oligonucleotide greatly enhances signal. We conclude that non-radioactive TRF assays can be as efficient as radioactive methods in detecting and measuring telomere length in plants, making this assay suitable for medical and research laboratories unable to utilize radioactivity due to hazardous waste disposal and safety concerns.