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公司名称: New England Biolabs
产品编号: R0155L
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Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths
Author:
Date:
2015-12-05
[Abstract]  Chromosome ends - telomeres - are a focus of intensive research due to their importance for the maintenance of chromosome stability. Their shortening due to incomplete replication functions as a molecular clock counting the number of cell divisions, and ultimately results in cell-cycle arrest and cellular senescence. Determination of telomere lengths is an essential approach in telomere biology for research and diagnostic applications. Terminal Restriction Fragments (TRF) analysis is the oldest approach to analyze telomere lengths and remains the “gold standard” even in current studies. This ... [摘要]  染色体末端 - 端粒是密集研究的焦点,因为它们对维持染色体稳定性的重要性。它们由于不完全复制而缩短作为计数细胞分裂数目的分子时钟,最终导致细胞周期停滞和细胞衰老。端粒长度的测定是端粒生物学中用于研究和诊断应用的基本方法。末端限制性片段(TRF)分析是分析端粒长度的最古老的方法,并且即使在目前的研究中仍然是"金标准"。该技术依赖于重复的小卫星端粒单元不含有限制酶的靶位点的事实。因此,端粒保持相对长的片段(TRF),而基因组DNA被消化成短片段。然后通过与放射性标记的端粒探针杂交显现端粒DNA的片段。由于TRF除了端粒外还包括直到第一限制性位点的端粒相关DNA的短区域,结果稍微偏向更高的TRF值。因此,建议使用频繁的刀具或其混合物,以尽量减少这种差异。此外,通过使用TRF分析,可以区分真正(末端)端粒与间质端粒重复(ITR)(Richards和Ausubel,1988)。在该方法中,首先将BAL31消化应用于高分子量DNA。酶从其末端逐渐降解线性DNA。然后用一种或多种限制酶消化降解的DNA,并通过凝胶电泳分离片段。印迹后,用末端标记序列或端粒序列探测膜。真正的TRF可以区别于ITR,因为它们随着BAL31消化时间的增加而逐步缩短,而ITR是BAL31抗性的。在时间零时的TRF BAL31消化模式表示近似端粒长度(Fajkus等人,2005)。

Telomere Restriction Fragment (TRF) Analysis
Author:
Date:
2015-11-20
[Abstract]  While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive ... [摘要]  虽然端粒酶在约90%的原发性人类肿瘤中表达,但除了瞬时增殖的干细胞样细胞之外,大多数体细胞组织细胞不具有可检测的端粒酶活性(Shay和Wright,1996; Shay和Wright,2001)。由于末端复制(滞后链合成)问题和其它原因例如氧化损伤,端粒在正常细胞中的每个细胞分裂(包括增殖的干细胞样细胞)逐渐缩短,因此所有体细胞具有有限的细胞增殖能力(Hayflick极限) (Hayflick和Moorhead,1961; Olovnikov,1973)。渐进性端粒缩短最终导致正常细胞中的生长停滞,其被称为复制衰老(Shay等人,1991)。一旦端粒酶在癌细胞中被激活,通过在染色体末端添加TTAGGG重复来稳定端粒长度,从而使细胞分裂无限延续(Shay和Wright,1996; Shay和Wright,2001)。因此,衰老和癌症之间的联系可以部分地解释端粒生物学。有许多快速和方便的方法来研究端粒生物学,例如端粒限制性片段(TRF),端粒重复扩增方案(Telomere Repeat Amplification Protocol, TRAP)(Mender and Shay,2015b)和端粒功能障碍诱导Foci(TIF)分析 ...

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