Telomere length measurement by a novel monochrome multiplex quantitative PCR method.
Telomere length measurement by a novel monochrome multiplex quantitative PCR method.
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DOI：
10.1093/nar/gkn1027
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发表时间：
2009-02
影响因子：
14.9
通讯作者：
Cawthon RM
中科院分区：
文献类型：
--
作者：
Cawthon RM
目前端粒长度的定量聚合酶链反应（QPCR）测定法测量一组反应威尔斯孔中实验DNA样品中的端粒（T）信号，以及与参考DNA相比，单独威尔斯孔中的单拷贝基因（S）信号，以产生与平均端粒长度成比例的相对T/S比。多路复用该测定是期望的，因为移液的DNA量的变化将不再有助于T/S的变化，因为T和S将在每个反应内从相同的输入DNA收集。多路复用还增加了吞吐量并降低了成本，因为需要的反应数量减少了一半。在这里，我们提出了第一个用于端粒长度测量的多重QPCR方法。值得注意的是，在该系统中，单个荧光DNA嵌入染料就足够了，因为T信号可以在S信号上升到基线以上之前的早期循环中收集，并且S信号可以在完全熔化端粒产物的温度下收集，将其信号发送到基线。与我们原来的单重方法（R2 = 0.677）相比，该单色多重QPCR方法（R2 = 0.844）的T/S比值与Southern印迹法测量的末端限制性片段（TRF）长度的相关性更强。不同日期独立运行的多重T/S结果具有高度重现性（R2 = 0.91）。
The current quantitative polymerase chain reaction (QPCR) assay of telomere length measures telomere (T) signals in experimental DNA samples in one set of reaction wells, and single copy gene (S) signals in separate wells, in comparison to a reference DNA, to yield relative T/S ratios that are proportional to average telomere length. Multiplexing this assay is desirable, because variation in the amount of DNA pipetted would no longer contribute to variation in T/S, since T and S would be collected within each reaction, from the same input DNA. Multiplexing also increases throughput and lowers costs, since half as many reactions are needed. Here, we present the first multiplexed QPCR method for telomere length measurement. Remarkably, a single fluorescent DNA-intercalating dye is sufficient in this system, because T signals can be collected in early cycles, before S signals rise above baseline, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation of T/S ratios with Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this monochrome multiplex QPCR method (R2 = 0.844) than with our original singleplex method (R2 = 0.677). Multiplex T/S results from independent runs on different days were highly reproducible (R2 = 0.91).
影响因子：
14.9
作者：
Cawthon, RM
通讯作者：
Cawthon, RM
DOI：
10.1016/s0167-4781(00)00200-1
发表时间：
2000-11-15
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION
影响因子：
--
作者：
Kainz, P
通讯作者：
Kainz, P
DOI：
10.1073/pnas.86.1.232
发表时间：
1989-01-01
影响因子：
11.1
作者：
SHEFFIELD, VC;COX, DR;MYERS, RM
通讯作者：
MYERS, RM